Recombinant peptide production using a cross-linkable solubility tag

ABSTRACT

The invention relates to the recombinant expression of a peptide of interest in the form of a fusion protein comprising a solubility tag. The fusion protein comprises at least two portions separated by a cleavable peptide sequence wherein one portion is devoid of cysteine residues and the second portion comprises an effective number of cross-linkable cysteine residues. After cell lysis and isolation of the fusion protein, the fusion protein is subsequently cleaved into a mixture of first and second portions. Oxidative cross-linking is used to selectively precipitate one of the two portions to facilitate simple and effective separation of the peptide of interest.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/172,395 filed Jul. 14, 2008, now pending, which claims the benefit ofU.S. Provisional Application No. 60/951,754 filed Jul. 25, 2007, nowexpired.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology, microbiology,and recombinant production of fusion peptides. More specifically, aprocess to obtain a peptide of interest from a mixture of peptidefragments produced by the cleavage of a fusion peptide is provided.

BACKGROUND OF THE INVENTION

Proteins and peptides are polymers of amino acids that have a widevariety of uses. Peptides are characteristically distinguished fromproteins by their smaller size and their lack of tertiary structureneeded for complex functionality, such as enzymatic activity. Syntheticpeptides that can be designed to exhibit desirable and valuablecharacteristics have been developed for a variety of purposes.

The efficient production of bioactive proteins and peptides has become ahallmark of the biomedical and industrial biochemical industry.Bioactive peptides and proteins are used as curative agents in a varietyof diseases such as diabetes (insulin), viral infections and leukemia(interferon), diseases of the immune system (interleukins), and redblood cell deficiencies (erythropoietin) to name a few. Additionally,large quantities of proteins and peptides are needed for variousindustrial applications including, for example, the pulp and paper andpulp industries, textiles, food industries, sugar refining, wastewatertreatment, production of alcoholic beverages and as catalysts for thegeneration of new pharmaceuticals.

With the advent of the discovery and implementation of combinatorialpeptide screening technologies such as bacterial display (Kemp, D. J.;Proc. Natl. Acad. Sci. USA 78(7): 4520-4524 (1981); yeast display (Chienet al., Proc Natl Acad Sci USA 88(21): 9578-82 (1991)), combinatorialsolid phase peptide synthesis (U.S. Pat. No. 5,449,754; U.S. Pat. No.5,480,971; U.S. Pat. No. 5,585,275 and U.S. Pat. No. 5,639,603), phagedisplay technology (U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484;U.S. Pat. No. 5,571,698; and U.S. Pat. No. 5,837,500), ribosome display(U.S. Pat. No. 5,643,768; U.S. Pat. No. 5,658,754; and U.S. Pat. No.7,074,557), and mRNA display technology (PROFUSION™; U.S. Pat. No.6,258,558; U.S. Pat. No. 6,518,018; U.S. Pat. No. 6,281,344; U.S. Pat.No. 6,214,553; U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,207,446; U.S.Pat. No. 6,846,655; U.S. Pat. No. 6,312,927; U.S. Pat. No. 6,602,685;U.S. Pat. No. 6,416,950; U.S. Pat. No. 6,429,300; U.S. Pat. No.7,078,197; and U.S. Pat. No. 6,436,665) new applications for peptideshaving specific binding affinities have been developed. In particular,peptides are being looked to as linkers in biomedical fields for theattachment of diagnostic and pharmaceutical agents to surfaces (seeGrinstaff et al, U.S. Patent Application Publication No. 2003/0185870and Linter in U.S. Pat. No. 6,620,419), as well as in the personal careindustry for the attachment of benefit agents to body surfaces such ashair and skin (see commonly owned U.S. patent application Ser. No.10/935,642, and Janssen et al. U.S. Patent Application Publication No.2003/0152976), and in the printing industry for the attachment ofpigments to print media (see commonly owned U.S. patent application Ser.No. 10/935,254).

In some cases commercially useful proteins and peptides may besynthetically generated or isolated from natural sources. However, thesemethods are often expensive, time consuming and characterized by limitedproduction capacity. The preferred method of protein and peptideproduction is through the fermentation of recombinantly constructedorganisms, engineered to over-express the protein or peptide ofinterest. Although preferable to synthesis or isolation, recombinantexpression of peptides has a number of obstacles to be overcome in orderto be a cost-effective means of production. For example, peptides (andin particular short peptides) produced in a cellular environment areoften soluble and susceptible to degradation from the action of nativecellular proteases. Purification can be difficult, resulting in pooryields depending on the nature of the protein or peptide of interest.

One means to mitigate the above difficulties is the use the geneticchimera for protein and peptide expression. A chimeric protein or“fusion protein” is a polypeptide comprising at least one portion of thedesired protein product fused to at least one portion comprising apeptide tag. The peptide tag may be used to assist protein folding,assist post expression purification, protect the protein from the actionof degradative enzymes, and/or assist the protein in passing through thecell membrane.

In many cases it is useful to express a protein or peptide in insolubleform, particularly when the peptide of interest is rather short,substantially soluble, and subject to proteolytic degradation within thehost cell. Production of the peptide in insoluble form both facilitatessimple recovery and protects the peptide from the undesirableproteolytic degradation. One means to produce the peptide in insolubleform is to recombinantly produce the peptide of interest in the form ofan insoluble fusion protein by including within the fusion construct atleast one solubility tag (i.e., an inclusion body tag) that promotesinclusion body formation. Typically, the fusion protein is also designedto include at least one cleavable peptide linker so that the peptide ofinterest can be subsequently recovered from the fusion protein. Thefusion protein may be designed to include a plurality of inclusion bodytags, cleavable peptide linkers, and regions encoding the peptide ofinterest.

Fusion proteins comprising a peptide tag that facilitate the expressionof insoluble proteins are well known in the art. Typically, the tagportion of the chimeric or fusion protein is large, increasing thelikelihood that the fusion protein will be insoluble. Example of largepeptide tags typically used include, but are not limited tochloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem.,174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. PeptideProtein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)),glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) andHancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S.Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614(1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology,12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos etal., J. Am. Chem. Soc. 116:4599 (1994) and co-owned U.S. PatentPublication No. 2006/0222609), ubiquitin (Pilon et al., Biotechnol.Prog., 13:374-79 (1997), bovine prochymosin (Naught et al., Biotechnol.Bioengineer. 57:55-61 (1998), and bactericidal/permeability-increasingprotein (“BPI”; Better, M. D. and Gavit, P D., U.S. Pat. No. 6,242,219).The art is replete with specific examples of this technology, see forexample U.S. Pat. No. 6,613,548, describing fusion protein ofproteinaceous tag and a soluble protein and subsequent purification fromcell lysate; U.S. Pat. No. 6,037,145, teaching a tag that protects theexpressed chimeric protein from a specific protease; U.S. Pat. No.5,648,244, teaching the synthesis of a fusion protein having a tag and acleavable linker for facile purification of the desired protein; andU.S. Pat. No. 5,215,896; U.S. Pat. No. 5,302,526; U.S. Pat. No.5,330,902; and U.S. Patent Publication No. 2005/221444, describingfusion tags containing amino acid compositions specifically designed toincrease insolubility of the chimeric protein or peptide.

Although the above methods are useful for the expression of fusionproteins, they often incorporate large inclusion body tags that decreasethe potential yield of desired peptide of interest. This is particularlyproblematic in situations where the desired protein or peptide is small.In such situations it is advantageous to use a small inclusion body tagto maximize yield.

Shorter inclusion tags have recently been developed from the Zea mayszein protein (co-pending U.S. patent application Ser. No. 11/641,936),the Daucus carota cystatin (co-pending U.S. patent application Ser. No.11/641,273), an amyloid-like hypothetical protein from Caenorhabditiselegans (co-owned U.S. patent application Ser. No. 11/516,362), and tagscomprising a n-sheet tape architecture (Aggeli et al., J. Amer. Chem.Soc., 125:9619-9628 (2003); Aggeli et al., PNAS, 98(21):11857-11862(2001); Aggeli et al., Nature, 386:259-262 (1997); Aggeli et al., J.Mater Chem, 7(7):1135-1145 (1997); and co-pending U.S. patentapplication Ser. No. 11/782,836. The use of short inclusion body tagsincreases the yield of the target peptide produced within therecombinant host cell.

Recovering the recombinantly produced peptide of interest from thefusion protein typically involves at least on cleavage step used toseparate the peptide of interest from the inclusion body tag. Oncecleaved, the peptide of interest is recovered from the mixture ofpeptide fragments. However, recovery of the peptide of interest is oftendifficult, especially when the inclusion body tag and the peptide ofinterest are similar in size and/or exhibit similar solubilitycharacteristics.

The problem to be solved is to provide a cost effective process toseparate the inclusion body tag from the peptide of interest.

SUMMARY OF THE INVENTION

The stated problem has been solved by providing a process to obtain apeptide of interest from a mixture of peptide fragments obtained aftercleaving the fusion peptide. Specifically, an effective number ofcross-linkable cysteine residues are engineered into only one of the twocomponents of the fusion peptide (i.e. the portion comprising theinclusion body tag or the portion comprising the peptide of interest).Cleavage of the fusion peptide forms a mixture of peptide fragments thatis subsequently subjected to oxidative conditions whereby intermolecularand intramolecular disulfide bonds are formed between the cysteineresidues engineered into only one of the two portions of the fusionpeptide. The selectively cross-linked peptide fragments are of highermolecular weight and are insoluble within the process matrix. Suitableprocess conditions are used to ensure that the portion of the fusionpeptide designed to be devoid of cysteine residues remains substantiallysoluble (after cleavage). The insoluble component is easily separatedfrom the soluble component using any number of well known separationtechniques such as centrifugation and/or filtration.

In one embodiment, the inclusion body tag comprises an effective numberof cross-linkable cysteine residues while no cysteine residues arepresent in the peptide of interest. As such, a process to obtain apeptide of interest from a fusion protein is provided comprising:

-   -   a) providing a population of fusion peptides comprising the        general structure:

IBT-CS-POI

or

POI-CS-IBT

-   -   -   wherein;        -   i) IBT is an inclusion body tag comprising an effective            number of cysteine residues;        -   ii) CS is a cleavage site; and        -   iii) POI is a peptide of interest that does not include a            cysteine residue;

    -   b) cleaving the population of fusion peptides at said cleavage        site whereby the inclusion body tag is no longer linked to the        peptide of interest and whereby a mixture of peptide molecules        is produced comprising a plurality of inclusion body tags and a        plurality of peptides of interest;

    -   c) subjecting the mixture of peptide molecules of step (b) to        oxidizing conditions whereby the inclusion body tags are        cross-linked; and

    -   d) recovering the peptide of interest.

In another embodiment, a method to obtain a peptide of interest is alsoprovided comprising:

-   -   a) providing a recombinant host cell comprising a nucleic acid        molecule encoding a fusion peptide comprising the general        structure:

IBT-CS-POI

or

POI-CS-IBT

-   -   -   wherein;        -   i) IBT is an inclusion body tag comprising an effective            number of cysteine residues;        -   ii) CS is a cleavage site; and        -   iii) POI is a peptide of interest that does not include a            cysteine residue;

    -   b) growing the host cell of step (a) under conditions whereby a        population of fusion peptides is produced;

    -   c) cleaving the population of fusion peptides at said cleavage        site whereby the inclusion body tag is no longer linked to the        peptide of interest and whereby a mixture of peptide molecules        is produced comprising a plurality of inclusion body tags and a        plurality of peptides of interest;

    -   d) subjecting the mixture of peptide molecules of step (c) to        oxidizing conditions whereby the inclusion body tags are        cross-linked; and

    -   e) recovering the peptide of interest.

The peptide of interest is isolated and/or recovered from the mixture ofpeptide molecules based on the difference in molecular weight and/orsolubility of the peptide of interest relative to the cross-linkedinclusion body tags. Recovery of the peptide of interest can use anynumber of well known separation techniques including, but not limited tocentrifugation and/or filtration (including microfiltration).

In an alternative embodiment, the peptide of interest comprises aneffective number of cross-linkable cysteine residues while the inclusionbody tag is devoid of cysteine residues. As such, a process to obtain apeptide of interest is provided comprising:

-   -   a) providing a population of fusion peptides comprising the        general structure:

IBT-CS-POI

or

POI-CS-IBT

-   -   -   wherein;        -   i) IBT is an inclusion body tag that does not include a            cysteine residue;        -   ii) CS is a cleavage site; and        -   iii) POI is a peptide of interest comprising an effective            number of cysteine residues;

    -   b) cleaving the population of fusion peptides at said cleavage        site whereby the inclusion body tag is no longer linked to the        peptide of interest and whereby a mixture of peptide molecules        is produced comprising a plurality of inclusion body tags and a        plurality of peptides of interest;

    -   c) subjecting the mixture of peptide molecules of step (b) to        oxidizing conditions whereby the peptides of interest are        cross-linked; and

    -   d) recovering the peptide of interest.

In yet another embodiment, a method to obtain a peptide of interest isalso provided comprising:

-   -   a) providing a recombinant host cell comprising a nucleic acid        molecule encoding a fusion peptide comprising the general        structure:

IBT-CS-POI

or

POI-CS-IBT

-   -   -   wherein;        -   i) IBT is an inclusion body tag that does not include a            cysteine residue;        -   ii) CS is a cleavage site; and        -   iii) POI is a peptide of interest comprising an effective            number of cysteine residues;

    -   b) growing the host cell of step (a) under conditions whereby a        population of fusion peptides is produced;

    -   c) cleaving the population fusion peptides at said cleavage site        whereby a mixture of peptide molecules is produce comprising a        plurality of inclusion body tags and a plurality of peptides of        interest;

    -   d) subjecting the mixture of peptide molecules of step (c) to        oxidizing conditions whereby the peptide of interest is        cross-linked; and

    -   e) recovering the peptide of interest.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of expression plasmid pLX121.

FIG. 2 is a diagram of expression plasmid pKSIC4-HC7723.

FIG. 3 is a diagram of expression plasmid pLR042.

FIG. 4 is a diagram of expression plasmid pLR186.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirementsfor Patent Applications Containing Nucleotide Sequences and/or AminoAcid Sequence Disclosures—the Sequence Rules”) and are consistent withWorld Intellectual Property Organization (WIPO) Standard ST.25 (1998)and the sequence listing requirements of the EPC and PCT (Rules 5.2 and49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions). The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleic acid sequence of plasmid pLX121.

SEQ ID NO: 2 is the nucleic acid sequence of plasmid pKSIC4-HC77623.

SEQ ID NO: 3 is the nucleic acid sequence of plasmid pLR042.

SEQ ID NO: 4 is the nucleic acid sequence of plasmid pLR186.

SEQ ID NO: 5 is the nucleic acid sequence encoding the KSI(4C) inclusionbody tag.

SEQ ID NO: 6 is the amino acid sequence of inclusion body tag KSI(4C).

SEQ ID NO: 7 is the nucleic acid sequence encoding the KSI.HC77607fusion peptide.

SEQ ID NO: 8 is the amino acid sequence of the KSI.HC77607 fusionpeptide.

SEQ ID NO: 9 is the amino acid sequence of hair binding domain KF11.

SEQ ID NO: 10 is the amino acid sequence of hair binding domain D21.

SEQ ID NO: 11 is the nucleic acid sequence encoding the peptide ofinterest HC77607 (multi-block hair-binding peptide).

SEQ ID NO: 12 is the amino acid sequence of HC77607 (multi-blockhair-binding peptide).

SEQ ID NO: 13 is the nucleic acid sequence encoding the KSI(C4).HC77643fusion peptide.

SEQ ID NO: 14 is the amino acid sequence of fusion peptideKSI(C4).HC77643.

SEQ ID NO: 15 is the amino acid sequence of hair binding peptide AO9.

SEQ ID NO: 16 is the nucleic acid sequence encoding the peptide ofinterest HC77643 (multi-block hair binding peptide).

SEQ ID NO: 17 is the amino acid sequence of the multi-block hair-bindingpeptide HC77643.

SEQ ID NO: 18 is the amino acid sequence of inclusion body tag IBT139.

SEQ ID NO: 19 is the nucleic acid sequence encoding fusion peptideIBT139.HC776124.

SEQ ID NO: 20 is the amino acid sequence of fusion peptideIBT139.HC776124.

SEQ ID NIO: 21 is the nucleic acid sequence encoding the peptide ofinterest HC776124 (multi-block hair-binding peptide).

SEQ ID NO: 22 is the amino acid sequence of multi-block hair-bindingpeptide HC776124.

SEQ ID NO: 23 is the nucleic acid sequence encoding inclusion body tagIBT186.

SEQ ID NO: 24 is the amino acid sequence of inclusion body tag IBT186.

SEQ ID NO: 25 is the nucleic acid sequence encoding the fusion peptideIBT186.HC776124.

SEQ ID NO: 26 is the amino acid sequence of fusion peptideIBT186.HC776124.

SEQ ID NO: 27 is the amino acid sequence of inclusion body tagIBT139.CCPGCC.

SEQ ID NO: 28 is the nucleic acid sequence encoding the fusion peptideIBT139.CCPGCC.HC776124.

SEQ ID NO: 29 is the amino acid sequence of fusion peptideIBT139.CCPGCC.HC776124.

SEQ ID NO: 30 is the amino acid sequence of a tetracysteine motif usefulas a cross-linkable tag.

SEQ ID NO: 31 is the nucleic acid sequence encoding the CCPGCCcross-linkable cysteine motif.

SEQ ID NO: 32 is the amino acid sequence of the CCPGCC cysteine motif.

SEQ ID NOs: 33-34 are the nucleic acid sequences of primers.

SEQ ID NOs: 35-37 and 43-58 are the amino acid sequences of hair bindingpeptides.

SEQ ID NOs: 38-42 are the amino acid sequences of peptides that bind toboth hair and skin.

SEQ ID NOs: 59-71 are the amino acid sequences of skin binding peptides.

SEQ ID NOs: 72-73 are the amino acid sequences of nail-binding peptides.

SEQ ID NOs: 74-102 are the amino acid sequences of antimicrobialpeptides.

SEQ ID NOs: 103-128 are the amino acid sequences of pigment bindingpeptides. Specifically, SEQ ID NOs: 103-106 bind to carbon black, SEQ IDNOs: 107-115 bind to CROMOPHTAL® yellow (Ciba Specialty Chemicals,Basel, Switzerland), SEQ ID NOs: 116-118 bind to SUNFAST® magenta (SunChemical Corp., Parsippany, N.J.), and SEQ ID NOs: 119-128 bind toSUNFAST® blue.

SEQ ID NOs: 129-134 are cellulose-binding peptides.

SEQ ID NOs: 135-162 are the amino acid sequences of polymer bindingpeptides. Specifically, SEQ ID NO: 135 binds to poly(ethyleneterephthalate), SEQ ID NOs: 136-147 bind to poly(methyl methacrylate),SEQ ID NOs: 148-153 bind to Nylon, and SEQ ID NOs: 154-162 bind topoly(tetrafluoroethylene).

SEQ ID NOs: 163-178 are the amino acid sequences of clay bindingpeptides.

SEQ ID NO: 179 is the amino acid sequence of the Caspase-3 cleavagesequence.

SEQ ID NO: 180 is the nucleic acid sequence of plasmid pLR435.

SEQ ID NO: 181 is the nucleic acid sequence encoding inclusion body tagIBT139(5C).

SEQ ID NO:182 is the amino acid sequence of inclusion body tagIBT139(5C).

SEQ ID NO: 183 is the nucleic acid sequence encoding fusion peptideIBT139(5C).HC776124.

SEQ ID NO: 184 is the amino acid sequence of fusion peptideIBT139(5C).HC776124.

SEQ ID NOs: 185-224 are the amino acid sequences of teeth-bindingpeptides (U.S. patent application Ser. No. 11/877,692).

DETAILED DESCRIPTION OF THE INVENTION

A process to obtain a peptide of interest from a fusion peptide isprovided. The peptide of interest is produced in the form of a fusionprotein engineered to have at least two functional portions separated byat least one cleavable peptide linker. One functional portion is asolubility tag (“inclusion body tag”) designed to promote production ofthe fusion protein in an insoluble form (i.e. in the form of inclusionbodies). Another portion of the fusion protein comprises the peptidetargeted for production (the “peptide of interest”). In a preferredembodiment, the fusion peptide is recombinantly produced in a microbialhost cell.

One of the two functional portions of the fusion protein is designed tohave an effective number of cross-linkable cysteine residues while theother functional portion is designed to be devoid of cysteine residues.The fusion protein is subjected to conditions whereby the peptide linkeris cleaved, forming a mixture of peptide fragments comprising theinclusion body tags and the peptides of interest. The mixture of peptidefragments is then subjected to oxidizing conditions whereby the portionof the fusion peptide designed having a plurality of cross-linkablecysteine residues is cross-linked by the formation of intermoleculardisulfide bonds. The cross-linked peptide molecules (higher in molecularweight and less soluble/insoluble) are separated from thenon-cross-linked soluble peptide molecules (i.e. the portion of thefusion peptide designed to be devoid of cross-linkable cysteineresidues). The cross-linked portion may be separated from thenon-cross-linked portion on the basis of molecular weight and/orsolubility. Methods to separate the two materials based on differencesin molecular weight and/or solubility are well known in the art and mayinclude, but are not limited to techniques such as centrifugation and/orfiltration.

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” refers to modifying the quantity of aningredient or reactant of the invention or employed refers to variationin the numerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

As used herein, the terms “fusion protein”, “fusion peptide”, “chimericprotein”, and “chimeric peptide” will be used interchangeably and willrefer to a polymer of amino acids (peptide, oligopeptide, polypeptide,or protein) comprising at least two portions, each portion comprising adistinct function. One portion of the fusion peptide comprises at leastone inclusion body tag (IBT). The second portion comprises at least onepeptide of interest (POI). The fusion protein additionally includes atleast one cleavable peptide linker (CL) that facilitates cleavage(chemical and/or enzymatic) and separation of the inclusion body tag(s)and the peptide(s) of interest. The fusion protein is designed such thateither the inclusion body tag or the peptide of interest comprises aplurality (e.g., 3 or more) cross-linkable cysteine residues(cross-linkable cysteine residues). Once the fusion protein is cleaved(using acid cleavage and/or enzymatic cleavage), the portion comprisingthe inclusion body tag is separated from the portion comprising thepeptide of interest by selectively cross-linking the portion comprisingthe cross-linkable cysteine residues. Oxidative cross-linking can becarried out using any number of techniques (i.e. bubbling oxygen throughthe mixture and/or by the use of chemical oxidants). The cross-linkedportion is separated from portion devoid of cysteine residues using anynumber of simple separation techniques including, but not limited tocentrifugation, filtration, and combinations thereof.

As used herein, the term “effective number of cysteine residues” is usedto describe the number of cysteine residues required to obtain thedesired effect (i.e. the ability to use oxidative cross-linking toselectively cross-link at least one portion of the cleaved fusionpeptide). It is well within the skill of one in the art to vary thenumber and/or location of the cysteine residues within the fusionpeptide to practice the present process. In one embodiment, theeffective number of cysteine residues is at least 3, preferably at least4. In another embodiment, the effective number of cysteine residues is 3to about 20, preferably 3 to about 10, more preferably 3 to about 6,more preferably 3 to about 5, and most preferably 4 to 5 cross-linkablecysteine residues.

As used herein, the terms “inclusion body tag” and “solubility tag” areused interchangeably and will be abbreviated “IBT” and will refer apolypeptide that facilitates/promotes formation of inclusion bodies whenfused to a peptide of interest. The peptide of interest is typicallysoluble within the host cell and/or host cell lysate when not fused toan inclusion body tag. Fusion of the peptide of interest to theinclusion body tag produces an insoluble fusion protein that typicallyagglomerates into intracellular bodies (inclusion bodies) within thehost cell. In one embodiment, the fusion protein comprises at least oneportion comprising an inclusion body tag and at least one portioncomprising the polypeptide of interest. In one embodiment, theprotein/peptide of interest is separated from the inclusion body tagusing at least one cleavable peptide linker elements (“cleavage sites”,abbreviated herein as “CS”).

As used herein, “cleavable linker elements”, “peptide linkers”, and“cleavable peptide linkers” will be used interchangeably and refer tocleavable peptide segments separating the inclusion body tag(s) and thepeptide(s) of interest. The cleavable peptide linker provides a sitewithin the fusion peptide for selective cleavage of the fusion peptide(i.e. the “cleavage site” or “cleavage sequence”). In one embodiment,the fusion peptide is designed to have at least one cleavable peptidelinker comprising a cleavage site separating the IBT from the POI. In apreferred embodiment, the arrangement of the cleavage site within thefusion protein comprises an arrangement of IBT-CS-POI wherein thecleavage site is at least one acid labile DP moiety. In one embodiment,the fusion peptide comprises a plurality of POIs and/or a plurality ofIBTs separated by one or more cleavage sites so long as a firstfunctional portion (e.g. IBTs) can be selectively separated from asecond functional portion (e.g. POIs) using the present process ofoxidatively cross-linking an effective number of cysteine residuesincorporated into only one of the two functional portions of the fusionpeptide.

After the inclusion bodies are separated and/or partially-purified orpurified from the cell lysate, the cleavable linker elements can becleaved chemically and/or enzymatically to separate the inclusion bodytag from the peptide of interest. The cleavable peptide linker may befrom 1 to about 50 amino acids, preferably from 1 to about 20 aminoacids in length. An example of a cleavable peptide linker is provided bySEQ ID NO: 179 (Caspase-3 cleavage sequence). Any cleavable peptidelinker can be used so long as the amino acid composition of the cleavagesite does not adversely impact the present process. The cleavablepeptide linkers may be incorporated into the fusion protein using anynumber of techniques well known in the art.

As used herein, an “inclusion body” is an intracellular amorphousdeposit comprising aggregated protein found in the cytoplasm of a cell.Peptides of interest that are soluble with the host cell and/or celllysates can be fused to one or more inclusion body tags to facilitateformation of an insoluble fusion protein. In an alternative embodiment,the peptide of interest may be partially insoluble in the host cell, butproduced at relatively lows levels where significant inclusion bodyformation does not occur. As such, the formation of inclusion bodieswill increase protein yield and/or protect the peptide from proteolyticdegradation. Formation of the inclusion body facilitates purification ofthe fusion peptide from the cell lysate using techniques well known inthe art such as centrifugation and filtration. The fusion peptide(“chimeric peptide”) is designed to include one or more cleavablepeptide linkers (encoding a cleavage site) separating the portion(s)comprising the peptide(s) of interest from the portion(s) comprising theinclusion body tag(s). The cleavable peptide linker is designed so thatthe portion comprising the inclusion body tag and the portion comprisingthe peptide of interest can be separated by cleaving fusion peptide atthe desired cleavage site (CS). The cleavage site can be cleavedchemically (e.g., acid hydrolysis) or enzymatically (i.e., use of aprotease/peptidase that preferentially recognizes an amino acid cleavagesite and/or sequence within the cleavable peptide linker). Once thefusion peptide is cleaved, the inclusion body tag(s) can be separatedfrom the peptide of interest using the present process of selectivecross-linking.

As used herein, the terms “cross-linking”, “oxidative cross-linking”,and “cysteine cross-linking” refer the present process of cross-linkingthe thiol groups of cysteine residues (i.e. forming intermolecular andintramolecular disulfide bonds) under oxidizing conditions. Bydefinition, the formation of intermolecular disulfide bonds occursbetween two or more molecules (i.e. a “plurality”) comprising aneffective number cross-linkable cysteine residues. As used herein, a“plurality” of molecules will alternatively be referred to herein as a“population” of molecules. In order to promote intermolecularcross-linking, an effective number (i.e. a plurality of at least 3)cross-linkable cysteine residues are incorporated into either theportion comprising the inclusion body tag or the portion comprising thepeptide of interest. In one embodiment, at least 3 cysteine residues areincorporated into the portion of the fusion protein targeted forcross-linking, preferably 3 to about 20 cysteine residues, morepreferably 3 to about 10 cysteine residues, yet even more preferably 3to about 6 cysteine residues, more preferably 3 to about 5 cysteineresidues, and most preferably about 4 or 5 cysteine residues are used.In a preferred embodiment, the cross-linkable cysteine residues areengineered into the inclusion body tag so that the peptide of interest(which is this case would not contain a cross-linkable cysteine residue)is isolated as a soluble peptide from the insoluble, cross-linked,inclusion body tags. In another embodiment, the cross-linkable cysteineresidues are incorporated into the peptide of interest while the portioncomprising the inclusion body tag does not include any cross-linkablecysteine residues. When the peptide of interest is separated from theinclusion body tag as a cross-linked peptide agglomerate (typicallyinsoluble), the cross-linked peptide of interest may subsequently besubjected to reducing conditions prior to preparing commercialformulations using the peptide of interest.

As used herein, the term “oxidizing conditions” refers to reactionconditions which favor and promote the formation of disulfide bondsbetween cysteine residues. Disulfide bond formation can be induced byany number of means well known in the art including, but not limited tocontacting the cross-linkable cysteine residues with a gas comprised ofoxygen (i.e. diatomic [O₂] and/or triatomic oxygen [O₃]) and/or theaddition of chemical oxidants. The use of gas comprising molecularoxygen is preferred. In a further embodiment, a gas comprising diatomicand/or triatomic oxygen is bubbled and/or sparged through the aqueousreaction solution for a period of time to achieve effective oxidativecross-linking. The oxidative cross-linking step may optionally includethe act of mixing and/or stirring of the aqueous reaction mixture foroptimal results. Examples of chemical oxidants are well-known in the artand may include, but are not limited to peroxide compounds,hypochlorite, halogens, and permanganate salts; to name a few.

As used herein, the term “reducing conditions” refers to reactionconditions which favor and promote the reduction of disulfide bondsbetween cysteine residues (i.e. breaks disulfide bond used forcross-linking). Disulfide bonds can be reduced by any number of meanswell known such as the use of nitrogen purge and/or a chemical reducingagent such as Na₂SO₃, DTT (dithiothreitol), TCEP(tris(2-carboxyethyl)phosphine), 2-mercaptoethanol,2-mercaptoethylamine, and mixtures thereof. Generally reducing agentsinclude those that contain thiol groups, those that are phosphines andtheir derivatives as well as sulfites and thiosulfites.

As used herein, the term “solubility” refers to the amount of asubstance that can be dissolved in a unit volume of a liquid underspecified conditions. As used herein, the term is used to describe theability of a peptide (inclusion body tag, peptide of interest, or fusionpeptide) to be suspended in a volume of solvent, such as a biologicalbuffer. In one embodiment, the peptides targeted for production(“peptides of interest”) are normally soluble in the cell and/or celllysate under normal physiological conditions. Fusion of one or moreinclusion body tags (IBTs) to the target peptide results in theformation of a fusion peptide that is insoluble under normalphysiological conditions, resulting in the formation of inclusionbodies. In the present process, the insoluble fusion peptides arerecovered from the cell and cleaved at the cleavage site into a mixtureof peptides fragments comprising a plurality of inclusion body tags anda plurality of peptide of interests. In one embodiment, the isolatedfusion peptide is solubilized prior to the introducing conditions thatpromote cleavage of the cleavable peptide linker. The mixture of peptideobtained after cleavage is then subjected to oxidizing conditionswhereby the peptide fragments comprising an effective number ofcross-linkable cysteine residues are selectively cross-linked intohigher molecular weight molecules that are typically insoluble under thechosen conditions while the non-cross-linked fragments remainsubstantially soluble.

As used herein, the term “pigment” refers to an insoluble, organic orinorganic colorant.

As used herein, the term “hair” as used herein refers to mammalian orhuman hair, eyebrows, and eyelashes.

As used herein, “HBP” means hair-binding peptide. As used herein, theterm “hair-binding peptide” refers to peptide sequences that bind withhigh affinity to hair. Examples of hair binding peptides have beenreported (U.S. patent application Ser. No. 11/074,473 to Huang et al.;WO 0179479; U.S. Patent Application Publication No. 2002/0098524 toMurray et al.; Janssen et al., U.S. Patent Application Publication No.2003/0152976 to Janssen et al.; WO 2004048399; U.S. application Ser. No.11/512,910, and U.S. patent application Ser. No. 11/696,380).Hair-binding peptides may include one or more hair-binding domains. Asused herein, hair-binding peptides comprising of a plurality ofhair-binding domains are referred to herein as “multi-block” or“multi-copy” hair-binding peptides. Examples of hair-binding peptidesare provided as SEQ ID NOs: 9-10, 12, 15, 17, 22, and 35-58 (Table 1).

As used herein, the term “skin” as used herein refers to mammalian orhuman skin, or substitutes for human skin, such as pig skin, VITRO-SKIN®(Innovative Measurement Solutions Inc., Milford, Conn.) and EPIDERM™(MatTek Corporation, Ashland, Mass.). Skin, as used herein, will referto a body surface generally comprising a layer of epithelial cells andmay additionally comprise a layer of endothelial cells.

As used herein, “SBP” means skin-binding peptide. As used herein, theterm “skin-binding peptide” refers to peptide sequences that bind withhigh affinity to skin. Examples of skin binding peptides have also beenreported (U.S. patent application Ser. No. 11/069,858 toBuseman-Williams; Rothe et. al., WO 2004/000257; and U.S. patentapplication Ser. No. 11/696,380). Examples of skin-binding peptides areprovided as SEQ ID NOs: 38-42 and 59-71 (Table 1).

As used herein, the term “nails” as used herein refers to mammalian orhuman fingernails and toenails.

As used herein, “NBP” means nail-binding peptide. As used herein, theterm “nail-binding peptide” refers to peptide sequences that bind withhigh affinity to the surface of fingernail or toenail tissue. Examplesof nail binding peptides have been reported (U.S. patent applicationSer. No. 11/696,380). Examples of nail-binding peptides are provided asSEQ ID NOs: 72-73 (Table 1).

As used herein, “TBP” means tooth-binding peptide. A tooth-bindingpeptide is a peptide that binds with high affinity to a mammalian orhuman tooth surface.

The term “tooth surface” will refer to a surface comprised of toothenamel (typically exposed after professional cleaning or polishing) ortooth pellicle (an acquired surface comprising salivary glycoproteins).Hydroxyapatite can be coated with salivary glycoproteins to mimic anatural tooth pellicle surface (tooth enamel is predominantly comprisedof hydroxyapatite).

As used herein, the terms “pellicle” and “tooth pellicle” will refer tothe thin film (typically ranging from about 1 μm to about 200 μm thick)derived from salivary glycoproteins which forms over the surface of thetooth crown. Daily tooth brushing tends to only remove a portion of thepellicle surface while abrasive tooth cleaning and/or polishing(typically by a dental professional) will exposure more of the toothenamel surface.

As used herein, the terms “enamel” and “tooth enamel” will refer to thehighly mineralized tissue which forms the outer layer of the tooth. Theenamel layer is composed primarily of crystalline calcium phosphate(i.e. hydroxyapatite; Ca₅(PO₄)₃OH) along with water and some organicmaterial. In one embodiment, the tooth surface is selected from thegroup consisting of tooth enamel and tooth pellicle.

As used herein, the term “tooth-binding peptide” will refer to a peptidethat binds to tooth enamel or tooth pellicle. In one embodiment, thetooth-binding peptides are from about 7 amino acids to about 50 aminoacids in length, more preferably, from about 7 amino acids to about 25amino acids in length, most preferably from about 7 to about 20 aminoacids in length. In a preferred embodiment, the tooth-binding peptidesare combinatorially-generated peptides.

Examples of tooth-binding peptides having been disclosed in co-pendingand co-owned U.S. patent application Ser. No. 11/877,692 and areprovided in Table 1. In a preferred embodiment, the tooth-bindingpeptide is selected from the group consisting of SEQ ID NOs: 185-224.

As used herein, “PBP” means polymer-binding peptide. As used herein, theterm “polymer-binding peptide” refers to peptide sequences that bindwith high affinity to a specific polymer (U.S. patent application Ser.No. 11/516,362). Examples include peptides that bind to poly(ethyleneterephthalate) (SEQ ID NO: 135), poly(methyl methacrylate) (SEQ IDNOs:136-147), Nylon (SEQ ID NOs: 148-153), and poly(tetrafluoroethylene)(SEQ ID NOs: 154-162).

As used herein, an “antimicrobial peptide” is a peptide having theability to kill microbial cell populations (U.S. patent application Ser.No. 11/516,362). Examples of antimicrobial peptides are provided as SEQID NOs: 74-102.

As used herein, “cellulose-binding peptide” refers to a peptide thatbinds with high affinity to cellulose. Examples of cellulose-bindingpeptides are provided as SEQ ID NOs: 129-134.

As used herein, “clay-binding peptide” refers to a peptide that bindswith high affinity to clay (U.S. patent application Ser. No.11/696,380). Examples of clay-binding peptides are provided as SEQ IDNOs: 163-178.

As used herein, the “benefit agent” refers to a molecule that imparts adesired functionality to a peptide complex involving the peptide ofinterest for a defined application. The benefit agent may be the peptideof interest itself or may be one or more molecules bound to (covalentlyor non-covalently), or associated with, the peptide of interest whereinthe binding affinity of the polypeptide is used to selectively targetthe benefit agent to the targeted material. In another embodiment, thetargeted polypeptide comprises at least one region having an affinityfor at least one target material (e.g., polymers, biological molecules,hair, skin, nail, teeth, other biological surfaces, other peptides,etc.) and at least one region having an affinity for the benefit agent(e.g., pharmaceutical agents, particulate benefit agents, clays, calciumcarbonate, pigments, conditioners, dyes, fragrances, and polymericcoatings applied to particulate benefit agents). In another embodiment,the peptide of interest comprises a plurality of regions having anaffinity for the target material and a plurality of regions having anaffinity for the benefit agent. In yet another embodiment, the peptideof interest comprises at least one region having an affinity for atargeted material and a plurality of regions having an affinity for avariety of benefit agents wherein the benefit agents may be the same ofdifferent. Examples of benefits agents may include, but are not limitedto conditioners for personal care products, particulate benefit agents(e.g. clays), pigments, dyes, fragrances, pharmaceutical agents (e.g.,targeted delivery of disease treatment agents), diagnostic/labelingagents, ultraviolet light blocking agents (i.e., active agents insunscreen protectants), and antimicrobial agents (e.g., antimicrobialpeptides), to name a few.

As used herein, the term “isolated nucleic acid molecule” is a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid molecule in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). In a furtherembodiment, the definition of “operably linked” may also be extended todescribe the products of chimeric genes, such as fusion proteins. Assuch, “operably linked” or “linked” will also refer to the linking of aninclusion body tag to a peptide of interest to be produced andrecovered. The inclusion body tag is “operably linked” to the peptide ofinterest if upon expression the fusion protein is insoluble andaccumulates in inclusion bodies in the expressing host cell. In apreferred embodiment, the fusion peptide will include at least onecleavable peptide linker useful in separating the inclusion body tagfrom the peptide of interest. In a further preferred embodiment, thecleavable linker is an acid cleavable aspartic acid-proline dipeptide(D-P) moiety. The cleavable peptide linkers may be incorporated into thefusion proteins using any number of techniques well known in the art.

As used herein, the terms “polypeptide” and “peptide” will be usedinterchangeably to refer to a polymer of two or more amino acids joinedtogether by a peptide bond, wherein the peptide is of unspecifiedlength, thus, peptides, oligopeptides, polypeptides, and proteins areincluded within the present definition. In one aspect, this term alsoincludes post expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, peptides containing one or moreanalogues of an amino acid or labeled amino acids and peptidomimetics.

As used herein, the terms “protein of interest”, “polypeptide ofinterest”, “peptide of interest”, “POI”, “targeted protein”, “targetedpolypeptide”, and “targeted peptide” will be used interchangeably andrefer to a protein, polypeptide, or peptide targeted for production thatis bioactive and may be expressed by the genetic machinery of a hostcell. In one embodiment, the peptide of interest comprises at least onebody surface-binding peptide selected from the group consisting ofhair-binding peptides, skin-binding peptides, nail-binding peptides, andteeth-binding peptides.

As used herein, the terms “bioactive”, “active”, and “peptide ofinterest activity” are used interchangeably and refer to the peptideshaving a defined activity, function, property or use making themdesirable for industrial/commercial applications. The bioactive peptidesmay be used in a variety of applications including, but not limited tocurative agents for diseases (e.g., insulin, interferon, interleukins,anti-angiogenic peptides (U.S. Pat. No. 6,815,426), and polypeptidesthat bind to defined cellular targets such as receptors, channels,lipids, cytosolic proteins, membrane proteins, peptides havingantimicrobial activity, peptides having an affinity for a particularmaterial (e.g., hair-binding peptides, skin-binding peptides,nail-binding peptides, teeth-binding peptides, cellulose-bindingpeptides, polymer-binding peptides, clay-binding peptides,silica-binding polypeptides, carbon nanotube binding polypeptides, andpeptides that have an affinity for particular animal or plant tissues)for targeted delivery of benefit agents. In one embodiment, the affinitypeptide is the benefit agent (e.g., the peptide of interest is aconditioning agent).

As used herein, the term “genetic construct” refers to a series ofcontiguous nucleic acids useful for modulating the genotype or phenotypeof an organism. Non-limiting examples of genetic constructs include butare not limited to a nucleic acid molecule, an open reading frame, agene, a plasmid, and the like.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid or asdefined Xaa X herein

As used herein, “gene” refers to a nucleic acid fragment that expressesa specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. “Synthetic genes” can beassembled from oligonucleotide building blocks that are chemicallysynthesized using procedures known to those skilled in the art. Thesebuilding blocks are ligated and annealed to form gene segments which arethen enzymatically assembled to construct the entire gene. “Chemicallysynthesized”, as related to a sequence of DNA, means that the componentnucleotides were assembled in vitro. Manual chemical synthesis of DNAmay be accomplished using well-established procedures, or automatedchemical synthesis can be performed using one of a number ofcommercially available machines. Accordingly, the genes can be tailoredfor optimal gene expression based on optimization of nucleotide sequenceto reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

Means to prepare the present peptides (inclusion body tags, cleavablepeptide linkers, cross-linkable cysteine moieties, peptides of interest,and fusion peptides) are well known in the art (see, for example,Stewart et al., Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill., 1984; Bodanszky, Principles of Peptide Synthesis,Springer-Verlag, New York, 1984; and Pennington et al., PeptideSynthesis Protocols, Humana Press, Totowa, N.J., 1994). The variouscomponents of the fusion peptides (inclusion body tag, peptide ofinterest, and the cleavable linker) described herein can be combinedusing carbodiimide coupling agents (see for example, Hermanson, Greg T.,Bioconiugate Techniques, Academic Press, New York (1996)), diacidchlorides, diisocyanates and other difunctional coupling reagents thatare reactive to terminal amine and/or carboxylic acid groups on thepeptides.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

Inclusion Body Tags

Fusion proteins comprising a protein tag (“inclusion body fusionpartner”) that facilitate the expression of insoluble proteins are wellknown in the art. The art typically uses inclusion body fusion partners(also referred to as “inclusion body tags” or “solubility tags”) thatare quite large, increasing the likelihood that the fusion protein willbe insoluble. Examples of large peptide tags typically used include, butare not limited to chloramphenicol acetyltransferase (Dykes et al., Eur.J. Biochem., 174:411 (1988), β-galactosidase (Schellenberger et al.,Int. J. Peptide Protein Res., 41:326 (1993); Shen et al., Proc. Nat.Acad. Sci. USA 281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)),glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) andHancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S.Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614(1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology,12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos etal., J. Am. Chem. Soc. 116:4599 (1994), ubiquitin (Pilon et al.,Biotechnol. Prog., 13:374-79 (1997), bovine prochymosin (Naught et al.,Biotechnol. Bioengineer. 57:55-61 (1998), andbactericidal/permeability-increasing protein (“BPI”; Better, M. D. andGavit, P D., U.S. Pat. No. 6,242,219). The art is replete with specificexamples of this technology, see for example U.S. Pat. No. 6,613,548,describing fusion protein of proteinaceous tag and a soluble protein andsubsequent purification from cell lysate; U.S. Pat. No. 6,037,145,teaching a tag that protects the expressed chimeric protein from aspecific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of afusion protein having a tag and a cleavable linker for facilepurification of the desired protein; and U.S. Pat. No. 5,215,896; U.S.Pat. No. 5,302,526; U.S. Pat. No. 5,330,902; and U.S. Patent applicationpublication No. 2005/221444, describing fusion tags containing aminoacid compositions specifically designed to increase insolubility of thechimeric protein or peptide.

Shorter inclusion tags have recently been developed from the Zea mayszein protein (co-pending U.S. patent application Ser. No. 11/641,936),the Daucus carota cystatin protein (co-pending U.S. patent applicationSer. No. 11/641,273), an amyloid-like hypothetical protein fromCaenorhabditis elegans (co-pending U.S. patent application Ser. No.11/516,362, and tags comprising a β-sheet tape architecture (Aggeli etal., J. Amer. Chem. Soc., 125:9619-9628 (2003); Aggeli et al., PNAS,98(21):11857-11862 (2001); Aggeli et al., Nature, 386:259-262 (1997);Aggeli et al., J. Mater Chem, 7(7):1135-1145 (1997); and co-pending U.S.patent application Ser. No. 11/782,836. The use of short inclusion bodytags increases the total amount of the target peptide produced (i.e.more of the fusion protein is the peptide of interest).

However, subsequent processing to separate the smaller inclusion bodytag from the peptide of interest is sometimes difficult, especially whenthe inclusion body tag and the peptide of interest have similarsolubility characteristics. As such, the present process provides a costeffective means to separate the inclusion body tag from the peptide ofinterest upon cleavage.

Inclusion Body Tags Comprising Cross-Linkable Cysteine Residues

The present method uses oxidative cross-linking to selectivelyprecipitate an inclusion body tag. The inclusion body tag generally hasan effective number of cross-linkable cysteine residues while thepeptide of interest is devoid of cross-linkable cysteine residues.

One of skill in the art can recombinantly engineer an effective numberof cross-linkable cysteine residues into the portion of the fusionprotein targeted for oxidative cross-linking. In one embodiment, theinclusion body tag comprises 3 or more cysteine residues, preferably 4or more cysteine residues, more preferably 3 to about 20, even morepreferably 3 to about 10, more preferably 3 to about 5, and mostpreferably 4 or 5 cross-linkable cysteine residues. The inclusion bodytags previously reported in the art that do not contain at least 3cysteine residues can be modified to include an effective amount ofcysteine residues to facilitate selective cross-linking. As such, anyinclusion body tag previously reported can be easily modified to includean effective number of cysteine residues using any number of well-knowntechniques known in the art of molecular biology. In another embodiment,previously reported inclusion body tags can be modified to comprise 3 ormore cysteine residues, preferably 4 or more cysteine residues, morepreferably 3 to about 20, even more preferably 3 to about 10, morepreferably 3 to about 5, and most preferably 4 or 5 cross-linkablecysteine residues.

In a preferred embodiment, the length of the inclusion body tag isminimized to increase the amount of the peptide of interest in thefusion protein. In one embodiment, the inclusion body tag comprising aneffective number of cross-linkable cysteine residues is less than 125amino acids in length, preferably less than 100 amino acids in length,more preferably less than 75 amino acids in length, even more preferablyless than 50 amino acids in lengths, yet even more preferably less than25 amino acids in length, and most preferably less than about 15 aminoacids in length. Means to identify small inclusion body tags have beenreported in the art (U.S. patent application Ser. No. 11/641,936, U.S.patent application Ser. No. 11/641,273, U.S. patent application Ser. No.11/641,981, and U.S. patent application Ser. No. 11/516,362).

The cysteine residues can be dispersed throughout the inclusion body tagand/or may be located on the amino and/or carboxy terminus of theinclusion body tag. In one embodiment, an effective number ofcross-linkable cysteine residues are added to a short inclusion body tag(e.g., no more than 125 amino acids in length). In another embodiment, across-linkable cysteine motif may also be incorporated into the portionof the fusion protein comprising the inclusion body tag to provide aneffective number of cross-linkable cysteine residues. When adding across-linkable cysteine motif to an inclusion body tag (i.e. one thatpreviously did not contain an effective number of cross-linkablecysteine residues), it is desirable to use a motif that is relativelyshort in order to minimize the impact on peptide yield. In oneembodiment, the cross-linkable cysteine motif is operably linked to theinclusion body tag and comprises 3 or more cysteine residues, preferably4 or more cysteine residues, more preferably 3 to about 20, even morepreferably 3 to about 10, more preferably 3 to about 5, and mostpreferably 4 or 5 cross-linkable cysteine residues wherein the additionof the cross-linkable cysteine motif provides and effective number ofcross-linkable cysteine residue to the inclusion body tag. In apreferred embodiment, the inclusion body tag is comprises thetetracysteine moiety (Cys-Cys-Xaa₁-Xaa₂-Cys-Cys; SEQ ID NO: 30) whereinXaa₁ and Xaa₂ is any amino acid. In a preferred embodiment, Xaa₁ is Proand Xaa₂ is Gly (Cys-Cys-Pro-Gly-Cys-Cys; SEQ ID NO: 32).

In one embodiment, the fusion peptide includes at least one cleavagesite (CS) useful in separating the peptide of interest from theinclusion body tag(s). In another embodiment, the cleavage site isprovided by a cleavable peptide linker. The CS can be an enzymaticcleavage sequence or a chemical cleavage sequence. In another preferredembodiment, the cleavable peptide linker comprises at least one acidcleavable aspartic acid-proline moiety (i.e. a “DP” acid cleavagemoiety).

Peptides of Interest (POIs) Comprising Cross-Linkable Cysteine Residues

The peptide of interest may contain (or be modified to contain) aneffective number of cross-linkable cysteine residues. In thisembodiment, the inclusion body tags are designed to be devoid of anycross-linkable cysteine residues. The cross-linkable cysteine residuesmay be dispersed throughout the peptide of interest or may beincorporated into the portion of the fusion protein comprising thepeptide of interest in the form of a cross-linkable cysteine moiety. Ina further embodiment, a cross-linkable cysteine moiety may also be addedto the amino or carboxy terminus of the peptide of interest (forexample, to provide an effective number of cross-linkable cysteineresidues to the peptide of interest) when the portion comprising theinclusion body tag is devoid of cysteine residues so long as theaddition of the cross-linkable cysteine moiety does not adversely impactthe activity/functionality of the peptide of interest. Means todetermine the impact of incorporating one or more additional cysteineresidues to the portion of the fusion protein encoding the peptide ofinterest are well known in the art and will depend upon the nature ofthe peptide of interest (e.g. enzymatic activity, binding affinity,etc.). One of skill in the art can compare the functionality of thecysteine-modified POI versus the unmodified version to determine theimpact on the desired functionality of the POI.

Expressible Peptides of Interest

The peptide of interest (“expressible peptide” or “POI”) targeted forproduction using the present method is one that is appreciably solublein the host cell and/or host cell liquid lysate under normalphysiological conditions. In a preferred aspect, the peptides ofinterest are generally short (<300 amino acids in length) and difficultto produce in sufficient amounts due to proteolytic degradation and/ordifficult to isolate due to their high solubility. Fusion of the peptideof interest to at least one inclusion body tag creates a fusion peptidethat is typically insoluble in the host cell and/or host cell lysateunder normal physiological conditions. Production of the peptide ofinterest is typically increased when expressed and accumulated in theform of an insoluble inclusion body as the peptide is generally moreprotected from proteolytic degradation. Furthermore, the insolublefusion protein (typically in the form of an inclusion body) can beeasily separated from the host cell lysate using centrifugation orfiltration.

Inclusion body tags can be used in a process to produce any peptide ofinterest that is (1) typically soluble in the cell and/or cell lysateunder typical physiological conditions and/or (2) those that can beproduced at significantly higher levels when expressed in the form of aninclusion body. In a preferred embodiment, the peptide of interest isappreciably soluble in the host cell and/or corresponding cell lysateunder normal physiological and/or process conditions.

The length of the peptide of interest may vary as long as (1) thepeptide is appreciably soluble in the host cell and/or cell lysate,and/or (2) the amount of the targeted peptide produced is increased whenexpressed in the form of an insoluble fusion peptide/inclusion body(i.e. expression in the form of a fusion protein protect the peptide ofinterest from proteolytic degradation). Typically the peptide ofinterest is less than 300 amino acids in length, preferably less than200 amino acids in length, preferably less than 150 amino acids inlength, more preferably less than 100 amino acids in length, even morepreferably less than 80 amino acids in length, and most preferably lessthan 50 amino acids in length.

The function of the peptide of interest is not limited by the presentmethod and may include, but is not limited to bioactive molecules suchas curative agents for diseases (e.g., insulin, interferon,interleukins, peptide hormones, anti-angiogenic peptides, and peptidesthat bind to and affect defined cellular targets such as receptors,channels, lipids, cytosolic proteins, and membrane proteins; see U.S.Pat. No. 6,696,089), peptides having an affinity for a particularmaterial (e.g., biological tissues, biological molecules, hair-bindingpeptides (U.S. Pat. No. 7,220,405; U.S. patent application Ser. No.11/074,473; WO 0179479; U.S. Patent Application Publication No.2002/0098524; U.S. Patent Application Publication No. 2003/0152976; WO04048399; U.S. patent application Ser. No. 11/512,910; and U.S. patentapplication Ser. No. 11/696,380), skin-binding peptides (U.S. Pat. No.7,220,405; U.S. patent application Ser. No. 11/069,858; WO 2004/000257;and U.S. patent application Ser. No. 11/696,380), nail-binding peptides(U.S. Pat. No. 7,220,405; U.S. patent application Ser. No. 11/696,380),teeth-binding peptide (U.S. patent application Ser. No. 11/877,692),cellulose-binding peptides, polymer-binding peptides (nylon-bindingpeptides (U.S. patent application Ser. No. 11/607,723);polytetrafluoroethylene-binding peptides (U.S. patent application Ser.No. 11/607,734); polyethylene-binding peptides (U.S. patent applicationSer. No. 11/607,672); polystyrene-binding peptides (U.S. patentapplication Ser. No. 11/607,673); polypropylene-binding peptides (U.S.patent application Ser. No. 11/607,792); polymethylmethacrylate-bindingpeptides (U.S. patent application Ser. No. 11/607,732)), clay bindingpeptides (U.S. patent application Ser. No. 11/696,380), silicon bindingpeptides, and carbon nanotube binding peptides (U.S. patent applicationSer. Nos. 11/093,533 and 11/093,873) for targeted delivery of at leastone benefit agent (see U.S. Pat. No. 7,220,405; U.S. patent applicationSer. No. 10/935,642; and U.S. patent application Ser. No. 11/074,473).

In one embodiment, the peptide of interest is selected from the groupconsisting of antimicrobial peptides (SEQ ID NOs: 74-102),polymer-binding peptides (SEQ ID NOs: 135-162), and the clay-bindingpeptides (SEQ ID NOs: (163-178).

Peptides of Interest—Body Surface-Binding Peptides: Hair-BindingPeptides, Nail-Binding Peptides, Skin-Binding Peptides, andTeeth-Binding Peptides

Hair-binding peptides (HBPs), nail-binding peptides (NBPs), skin-bindingpeptides (SBPs), and teeth-binding peptides (TBPs) as defined herein arepeptide sequences that bind with high affinity to hair, nail, skin orteeth; respectively. The hair-binding peptides, nail-binding peptides,skin-binding peptides, and teeth-binding peptides are typically fromabout 7 amino acids to about 100 amino acids in length, more preferablyabout 7 amino acids to about 50 amino acids in length, and mostpreferably about 7 to about 30 amino acids in length. Suitable hair-,nail-, skin-, and teeth-binding peptides may be selected using methodsthat are well known in the art or may be empirically generated

The hair-, nail-, skin- or teeth-binding peptides may be generatedrandomly and then selected against a specific hair, nail, skin, or toothsurface sample based upon their binding affinity for the substrate ofinterest, as described by Huang et al. in U.S. Patent ApplicationPublication No. 2005/0050656 or O'Brien et al. in U.S. patentapplication Ser. No. 11/877,692 or by a method using mRNA-display asdescribed in U.S. patent application Ser. No. 11/696,380, eachincorporated herein by reference. The generation of random libraries ofpeptides is well known and may be accomplished by a variety oftechniques including, bacterial display (Kemp, D. J.; Proc. Natl. Acad.Sci. USA 78(7):4520-4524 (1981), and Helfman et al., Proc. Natl. Acad.Sci. USA 80(1):31-35, (1983)), yeast display (Chien et al., Proc NatlAcad Sci USA 88(21):9578-82 (1991)), combinatorial solid phase peptidesynthesis (U.S. Pat. No. 5,449,754, U.S. Pat. No. 5,480,971, U.S. Pat.No. 5,585,275, U.S. Pat. No. 5,639,603), phage display technology (U.S.Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698,U.S. Pat. No. 5,837,500), ribosome display technology (U.S. Pat. No.5,643,768; U.S. Pat. No. 5,658,754; and U.S. Pat. No. 7,074,557), andmRNA display technology (U.S. Pat. No. 6,258,558; U.S. Pat. No.6,518,018; U.S. Pat. No. 6,281,344; U.S. Pat. No. 6,214,553; U.S. Pat.No. 6,261,804; U.S. Pat. No. 6,207,446; U.S. Pat. No. 6,846,655; U.S.Pat. No. 6,312,927; U.S. Pat. No. 6,602,685; U.S. Pat. No. 6,416,950;U.S. Pat. No. 6,429,300; U.S. Pat. No. 7,078,197; U.S. Pat. No.6,436,665; U.S. Pat. No. 6,361,943; and U.S. Pat. No. 6,228,994).

Any hair-binding, skin-binding, nail-binding or teeth-binding peptidemay be used, such as those reported in co-pending and commonly ownedU.S. Patent Application Publication No. 2005/0050656; U.S. PatentApplication Publication No. 2005/0226839, and U.S. patent applicationSer. No. 11/877,692; Estell et al. (WO 0179479); Murray et al., (U.S.Patent Application Publication No. 2002/0098524); Janssen et al., (U.S.Patent Application Publication No. 2003/0152976); Janssen et al., (WO04048399), O'Brien et al. (co-pending and commonly owned U.S. PatentApplication Publication No. 2006/0073111), Wang et al. (co-pending andcommonly owned U.S. patent application Ser. No. 11/359,163) and Wang etal. (co-pending and commonly owned U.S. patent application Ser. No.11/359,162), all of which are incorporated herein by reference.

In another preferred aspect, the hair-binding peptide is selected fromthe group consisting of SEQ ID NOs: 9, 10, 12, 15, 17, 22, and 35-58;the skin-binding peptide is selected from the group consisting of SEQ IDNOs: 38-42 and 59-71; the nail-binding peptide is selected from thegroup consisting of SEQ ID NOs: 72-73, and the teeth-binding peptides isselected from the group consisting of SEQ ID NOs: 185-224. In anotherembodiment, the peptide of interest is a non-naturally occurring peptideidentified from a combinatorially-generated library of peptides.

Alternatively, hair-, nail-, and skin-binding peptide sequences may alsobe generated empirically by designing peptides that comprise positivelycharged amino acids, which can bind to hair and skin via electrostaticinteraction, as described by Rothe et al. (WO 2004/000257). Theempirically generated hair, nail, and skin-binding peptides have betweenabout 7 amino acids to about 50 amino acids, and comprise at least about40 mole % positively charged amino acids, such as lysine, arginine, andhistidine. Peptide sequences containing tripeptide motifs such as HRK,RHK, HKR, RKH, KRH, KHR, HKX, KRX, RKX, HRX, KHX and RHX are mostpreferred where X can be any natural amino acid but is most preferablyselected from neutral side chain amino acids such as glycine, alanine,proline, leucine, isoleucine, valine and phenylalanine. In addition, itshould be understood that the peptide sequences must meet otherfunctional requirements in the end use including solubility, viscosityand compatibility with other components in a formulated product and willtherefore vary according to the needs of the application. In some casesthe peptide may contain up to 60 mole % of amino acids not comprisinghistidine, lysine or arginine. Suitable empirically generatedhair-binding, nail-binding, and skin-binding peptides include, but arenot limited to, SEQ ID NOs: 38-42 (see Table 1).

It may also be beneficial to use a mixture of different hair-binding,nail-binding, or skin-binding peptides. The peptides in the mixture needto be chosen so that there is no interaction between the peptides thatmitigates the beneficial effect. Suitable mixtures of hair-binding,nail-binding or skin-binding peptides may be determined by one skilledin the art using routine experimentation. Additionally, it may bedesirable to link two or more hair-binding peptides, nail-bindingpeptides or skin-binding peptides together, either directly or through aspacer, to enhance the interaction of the peptide to the substrate.Methods to prepare the multiple peptide compositions and suitablespacers are described below. Non-limiting examples are given in Table 1.

TABLE 1 Examples of Hair-Binding Peptides, Nail-Binding Peptides, Skin-Binding Peptides, and  Teeth-Binding Peptides SEQBody ID Surface NO: Sequence Hair  35 TPPELLHGDPRS (Shampoo Resistant)Hair   9 NTSQLST (also referred to (Shampoo herein as KF11) Resistant)Hair  10 RTNAADHP (also referred to herein as D21) Hair  36 RTNAADHPAAVTHair  15 IPWWNIRAPLNA (also referred to herein as AO9) Hair  37 DLTLPFHHair and  38 KRGRHKRPKRHK Skin (empirical) Hair and  39 RLLRLLR Skin(empirical) Hair and  40 HKPRGGRKKALH Skin (empirical) Hair and  41KPRPPHGKKHRPKHRPKK Skin (empirical) Hair and  42 RGRPKKGHGKRPGHRARK Skin(empirical) Hair (Multi-  12 GSDPNTSQLSTGGGRTNAA copy)DHPKCGGGNTSQLSTGGGR (also TNAADHPKCGGGNTSQLST referred to GGGRTNAADHPKCherein as “HC77607”) Hair (Multi-  43 PRTNAADHPAAVTGGGCGG copy)GRTNAADHPAAVTGGGCGG GRTNAADHPAAVTGGGC Hair (Multi-  44PRTNAADHPAAVTGGGCGG copy) GIPWWNIRAPLNAGGGCGG GDLTLPFHGGGC Hair (Multi- 45 PRTNAADHPGGGTPPELLHG copy) DPRSKCGGGRTNAADHPGG GTPPELLHGDPRSKCGGGRTNAADHPGGGTPPELLHGDP RSKC Hair (Multi-  46 PTPPTNVLMLATKGGGRTNA copy)ADHPKCGGGTPPTNVLMLAT KGGGRTNAADHPKCGGGTP PTNVLMLATKGGGRTNAADH PKCHair (Multi-  47 PRTNAADHPGGGTPPTNVLM copy) LATKKCGGGRTNAADHPGGGTPPTNVLMLATKKCGGGRT NAADHPGGGTPPTNVLMLAT KKC Hair (with  48TPPELLHGDPRSC cysteine at C-terminus) Hair  49 EQISGSLVAAPW Hair  50TDMQAPTKSYSN Hair  51 ALPRIANTWSPS Hair  52 LDTSFPPVPFHA Hair  53TPPTNVLMLATK (Shampoo Resistant) Hair  54 STLHKYKSQDPTPHH (ConditionerResistant) Hair  55 GMPAMHWIHPFA (Shampoo and Conditioner Resistant)Hair  56 HDHKNQKETHQRHAA (Shampoo and Conditioner Resistant) Hair  57HNHMQERYTDPQHSPSVNG (Shampoo L and Conditioner Resistant) Hair  58TAEIQSSKNPNPHPQRSWTN (Shampoo and Conditioner Resistant) Skin  59TPFHSPENAPGS Skin (Body  60 TMGFTAPRFPHY Wash Resistant) Skin (Body  61SVSVGMKPSPRP Wash Resistant) Skin (Body  62 NLQHSVGTSPVW Wash Resistant)Skin (Body  63 QLSYHAYPQANHHAP Wash Resistant) Skin (Body  64SGCHLVYDNGFCDH Wash Resistant) Skin (Body  65 ASCPSASHADPCAH WashResistant) Skin (Body  66 NLCDSARDSPRCKV Wash Resistant) Skin (Body  67NHSNWKTAADFL Wash Resistant) Skin (Body  68 SDTISRLHVSMT Wash Resistant)Skin (Body  69 SPYPSWSTPAGR Wash Resistant) Skin (Body  70DACSGNGHPNNCDR Wash Resistant) Skin (Body  71 DWCDTIIPGRTCHG WashResistant) Nail  72 ALPRIANTWSPS Nail  73 YPSFSPTYRPAF Tooth 185AHPESLGIKYALDGNSDPHA (pellicle) Tooth 186 ASVSNYPPIHHLATSNTTVN(pellicle) Tooth 187 DECMEPLNAAHCWR (pellicle) Tooth 188 DECMHGSDVEFCTS(pellicle) Tooth 189 DLCSMQMMNTGCHY (pellicle) Tooth 190 DLCSSPSTWGSCIR(pellicle) Tooth 191 DPNESNYENATTVSQPTRHL (pellicle) Tooth 192EPTHPTMRAQMHQSLRSSS (pellicle) P Tooth 193 GNTDTTPPNAVMEPTVQHK(pellicle) W Tooth 194 NGPDMVQSVGKHKNS (pellicle) Tooth 195NGPEVRQIPANFEKL (pellicle) Tooth 196 NNTSADNPPETDSKHHLSMS (pellicle)Tooth 197 NNTWPEGAGHTMPSTNIRQA (pellicle) Tooth 198 NPTATPHMKDPMHSNAHSS(pellicle) A Tooth 199 NPTDHIPANSTNSRVSKGNT (pellicle) Tooth 200NPTDSTHMMHARNHE (pellicle) Tooth 201 QHCITERLHPPCTK (pellicle) Tooth 202TPCAPASFNPHCSR (pellicle) Tooth 203 TPCATYPHFSGCRA (pellicle) Tooth 204WCTDFCTRSTPTSTSRSTTS (pellicle) Tooth 205 APPLKTYMQERELTMSQNKD (enamel)Tooth 206 EPPTRTRVNNHTVTVQAQQH (enamel) Tooth 207 GYCLRGDEPAVCSG(enamel) Tooth 208 LSSKDFGVTNTDQRTYDYTT (enamel) Tooth 209NFCETQLDLSVCTV (enamel) Tooth 210 NTCQPTKNATPCSA (enamel) Tooth 211PSEPERRDRNIAANAGRFNT (enamel) Tooth 212 THNMSHFPPSGHPKRTAT (enamel)Tooth 213 TTCPTMGTYHVCWL (enamel) Tooth 214 YCADHTPDPANPNKICGYSH(enamel) Tooth 215 AANPHTEWDRDAFQLAMPP (enamel) K Tooth 216DLHPMDPSNKRPDNPSDLHT (enamel) Tooth 217 ESCVSNALMNQCIY (enamel) Tooth218 HNKADSWDPDLPPHAGMSL (enamel) G Tooth 219 LNDQRKPGPPTMPTHSPAVG(enamel) Tooth 220 NTCATSPNSYTCSN (enamel) Tooth 221 SDCTAGLVPPLCAT(enamel) Tooth 222 TIESSQHSRTHQQNYGSTKT (enamel) Tooth 223VGTMKQHPTTTQPPRVSATN (enamel) Tooth 224 YSETPNDQKPNPHYKVSGTK (enamel)

Cleavable Peptide Linkers

The use of cleavable peptide linkers is well known in the art. Fusionpeptides comprising the present inclusion body tags will typicallyinclude at least one cleavable peptide sequence (i.e. cleavage site or“CS”) separating the inclusion body tag from the polypeptide ofinterest. The cleavable sequence facilitates separation of the inclusionbody tag(s) from the peptide(s) of interest. In one embodiment, thecleavable sequence may be provided by a portion of the inclusion bodytag and/or the peptide of interest (e.g., inclusion of an acid cleavableaspartic acid-proline moiety). In a preferred embodiment, the cleavablesequence is provided by including (in the fusion peptide) at least onecleavable peptide linker between the inclusion body tag and the peptideof interest.

Means to cleave the peptide linkers are well known in the art and mayinclude chemical hydrolysis, enzymatic cleavage agents, and combinationsthereof. In one embodiment, one or more chemically cleavable peptidelinkers are included in the fusion construct to facilitate recovery ofthe peptide of interest from the inclusion body fusion protein. Examplesof chemical cleavage reagents include cyanogen bromide (cleavesmethionine residues), N-chloro succinimide, iodobenzoic acid orBNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole] (cleavestryptophan residues), dilute acids (cleaves at aspartyl-prolyl bonds),and hydroxylamine (cleaves at asparagine-glycine bonds at pH 9.0); seeGavit, P. and Better, M., J. Biotechnol., 79:127-136 (2000); Szoka etal., DNA, 5(1):11-20 (1986); and Walker, J. M., The Proteomics ProtocolsHandbook, 2005, Humana Press, Totowa, N.J.)). In a preferred embodiment,one or more aspartic acid-proline acid cleavable recognition sites(i.e., a cleavable peptide linker comprising one or more D-P dipeptidemoieties) are included in the fusion protein construct to facilitateseparation of the inclusion body tag(s) form the peptide of interest. Inanother embodiment, the fusion peptide may include multiple regionsencoding peptides of interest separated by one or more cleavable peptidelinkers wherein the regions are separated by one or more cleavablepeptide linkers.

In another embodiment, one or more enzymatic cleavage sequences areincluded in the fusion protein construct to facilitate recovery of thepeptide of interest. Proteolytic enzymes and their respective cleavagesite specificities are well known in the art. In a preferred embodiment,the proteolytic enzyme is selected to specifically cleave only thepeptide linker separating the inclusion body tag and the peptide ofinterest. Examples of enzymes useful for cleaving the peptide linkerinclude, but are not limited to Arg-C proteinase, Asp-N endopeptidase,chymotrypsin, clostripain, enterokinase, Factor Xa, glutamylendopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, prolineendopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin,thrombin, trypsin, and members of the Caspase family of proteolyticenzymes (e.g. Caspases 1-10) (Walker, J. M., supra). An example of acleavage site sequence is provided by SEQ ID NO: 179 (Caspase-3 cleavagesite; Thornberry et al., J. Biol. Chem., 272:17907-17911 (1997) and Tyaset al., EMBO Reports, 1(3):266-270 (2000)).

Typically, the cleavage step occurs after the insoluble inclusion bodiesand/or insoluble fusion peptides have been separated from the celllysate. The cells can be lysed using any number of means well known inthe art (e.g. mechanical and/or chemical lysis). Methods to collectand/or isolate the insoluble inclusion bodies/fusion peptides from thecell lysate are well known in the art (e.g., centrifugation, filtration,and combinations thereof). Once recovered from the cell lysate, theinsoluble inclusion bodies and/or fusion peptides can be treated with acleavage agent (chemical or enzymatic) to cleavage the inclusion bodytag from the peptide of interest. In one embodiment, the fusion proteinand/or inclusion body is diluted and/or dissolved in a suitable solvent(e.g., water) prior to treatment with the cleavage agent. The cleavagestep is preferably conducted in an aqueous environment.

The inclusion body tag is separated from the peptide of interest usingoxidative cross-linking of cysteine residues incorporated into theinclusion body tag or the peptide of interest with the provision thatboth fragments cannot simultaneously contain an effective number ofcross-linkable cysteine residues. Cross-linking of the cysteine residuesunder oxidative conditions induces the formation of higher moleculeweight, insoluble protein agglomerates. The conditions are adjusted sothat the portion that does not contain the cross-linked cysteineresidues is appreciably soluble under the oxidizing conditions. As such,the portion of fusion protein comprising the inclusion body tag can beeasily and efficiently separated from the peptide of interest usingsimple separation techniques such as centrifugation and/or filtration.

In one embodiment, the peptide of interest is soluble while theinclusion body tag and/or fusion protein is insoluble in the definedprocess matrix (typically an aqueous matrix). In another embodiment, thepeptide of interest is insoluble while the inclusion body tag is solublein the defined process matrix. When the peptide on interest iscross-linked using the present process, an optional step may be added toreduce the cysteine cross-linking so that the peptide of interest can beisolated/purified in a monomeric and/or soluble form.

In an optional embodiment, the peptide of interest (once isolated afterthe present cross-linking step) may be further purified using any numberof well known purification techniques in the art such as ion exchange,gel purification techniques, and column chromatography (see U.S. Pat.No. 5,648,244), to name a few.

Fusion Peptides

The fusion peptide should include at least one inclusion body tag (IBT)operably linked to at least one peptide of interest. Typically, thefusion peptide includes at least one cleavable peptide linker having acleavage site between the inclusion body tag and the peptide ofinterest. In one embodiment, the inclusion body tag may include acleavage site whereby inclusion of a separate cleavable peptide linkermay not be necessary. In a preferred embodiment, the cleavage method ischosen to ensure that the peptide of interest is not adversely affectedby the cleavage agent(s) employed. In a further embodiment, the peptideof interest may be modified to eliminate possible cleavage sites (and/oramino acid residues sensitive to the cleavage agent) with the peptide solong as the desired activity of the peptide is not adversely affected.

One of skill in the art will recognize that the elements of the fusionprotein can be structured in a variety of ways. Typically, the fusionprotein will include at least one IBT, at least one peptide of interest(POI), and at least one cleavage site (CS; typically in the form of acleavable linker; CL) located between the IBT and the POI. The inclusionbody tag may be organized as a leader sequence or a terminator sequencerelative to the position of the peptide of interest within the fusionpeptide. In another embodiment, a plurality of IBTs, POIs, and cleavagesites are used when engineering the fusion peptide. In a furtherembodiment, the fusion peptide may include a plurality of IBTs (asdefined herein), POIs, and cleavage sites that are the same ordifferent.

The fusion peptide is typically insoluble in an aqueous matrix at atemperature of 10° C. to 50° C., preferably 10° C. to 40° C. undernormal physiological conditions. The aqueous matrix typically comprisesa pH range of 5 to 12, preferably 6 to 10, and most preferably 6 to 8.The temperature, pH, and/or ionic strength of the aqueous matrix can beadjusted to obtain the desired solubility characteristics of the fusionpeptide. For example, prior to acid cleavage, the conditions may beadjusted to solubilize the isolated fusion protein.

Method to Make a Peptide of Interest Using Insoluble Fusion Peptides

Chimeric genes are constructed using techniques well known in the art.The chimeric constructs are designed to encode at least one peptide ofinterest operably linked (via a cleavable peptide linker) to at leastone inclusion body tag. Expression of the chimeric genetic constructproduces an insoluble form of the peptide of interest that accumulatesin the form of inclusion bodies within the host cell. The host cell isgrown for a period of time sufficient for the insoluble fusion peptideto accumulate in the form of inclusion bodies within the cell.

The host cell is subsequently lysed using any number of techniques wellknown in the art. The insoluble fusion peptides/inclusion bodies arethen separated from the other components of the cell lysate using asimple and economical technique such as centrifugation and/or membranefiltration. The insoluble fusion peptide/inclusion body can then befurther processed in order to isolate the peptide of interest.Typically, this will include resuspension of the fusionpeptide/inclusion body in a liquid matrix suitable for cleaving thefusion peptide. The cleavage step can be conducted using any number oftechniques well known in the art (chemical cleavage, enzymatic cleavage,and combinations thereof) wherein acid cleavage is preferred.

After cleavage, the mixture of fusion peptide fragments is subjected tooxidative cross-linking whereby one of the components is selectivelycross-linked to facilitate separation. The cross-linked component isseparated from the soluble component(s) using any numbers of techniquesknown in the art. In a preferred embodiment, centrifugation and/orfiltration is used to separate the cross-linked material from thenon-cross-linked material.

Transformation and Expression

Recombinant expression of the chimeric genes encoding the desired fusionprotein can be prepared using techniques well known in the art.Typically, the chimeric constructs are engineered and expressed from avector transformed into an appropriate host cell. Typically, the vectoror cassette contains sequences directing transcription and translationof the relevant chimeric gene, a selectable marker, and sequencesallowing autonomous replication or chromosomal integration. Suitablevectors comprise a region 5′ of the gene which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host.

Initiation control regions or promoters, which are useful to driveexpression of the genetic constructs encoding the fusion peptide in thedesired host cell, are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving these constructs issuitable for the present invention including but not limited to CYC1,HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI (useful for expression in Saccharomyces); AOX1 (useful forexpression in Pichia); and lac, ara (pBAD), tet, trp, IP_(L), IP_(R),T7, tac, and trc (useful for expression in Escherichia coli) as well asthe amy, apr, npr promoters and various phage promoters useful forexpression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Preferred host cells for expression of the fusion peptide are microbialhosts that can be found broadly within the fungal or bacterial familiesand which grow over a wide range of temperature, pH values, and solventtolerances. For example, it is contemplated that any of bacteria, yeast,and filamentous fungi will be suitable hosts for expression of thenucleic acid molecules encoding fusion peptides. Because oftranscription, translation, and the protein biosynthetic apparatus isthe same irrespective of the cellular feedstock, genes are expressedirrespective of the carbon feedstock used to generate the cellularbiomass. Large-scale microbial growth and functional gene expression mayutilize a wide range of simple or complex carbohydrates, organic acidsand alcohols (i.e. methanol), saturated hydrocarbons such as methane orcarbon dioxide in the case of photosynthetic or chemoautotrophic hosts.However, the functional genes may be regulated, repressed or depressedby specific growth conditions, which may include the form and amount ofnitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrientincluding small inorganic ions. In addition, the regulation offunctional genes may be achieved by the presence or absence of specificregulatory molecules that are added to the culture and are not typicallyconsidered nutrient or energy sources. Growth rate may also be animportant regulatory factor in gene expression. Examples of host strainsinclude, but are not limited to fungal or yeast species such asAspergillus, Trichoderma, Saccharomyces, Pichia, Yarrowia, Candida,Hansenula, or bacterial species such as Salmonella, Bacillus,Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium,Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus,Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium,Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas,Methylomonas, Methylobacter, Methylococcus, Methylosinus,Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis,Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, andMyxococcus. Preferred bacterial host strains include Escherichia,Pseudomonas, and Bacillus. In a preferred aspect, the bacterial hoststrain is Escherichia coli.

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeast are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the expression of the present fusionpeptides.

Culture Conditions

Suitable culture conditions can be selected dependent upon the chosenproduction host. Typically, cells are grown at a temperature in therange of about 25° C. to about 40° C. in an appropriate medium. Suitablegrowth media in the present invention are common commercially preparedmedia such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth orYeast medium (YM) broth. Other defined or synthetic growth media mayalso be used and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science. The use of agents known to modulate cataboliterepression directly or indirectly, e.g., cyclic adenosine2′:3′-monophosphate, may also be incorporated into the fermentationmedium.

Suitable pH ranges for the fermentation are typically between pH 5.0 topH 9.0, where about pH 6.0 to about pH 8.0 is preferred.

Fermentations may be performed under aerobic or anaerobic conditionswherein aerobic conditions are preferred.

Process Steps Prior to Cysteine Cross-Linking

Recombinant production of fusion peptides/proteins in the form ofinclusion bodies is well known in the art. Typically, the recombinantcells (comprising the fusion protein) are homogenized to release theinsoluble inclusion bodies. Isolation of inclusion bodies from a celllysate are based on well known techniques including, but not limited tocentrifugation and/or filtration. The process typically involves severalcycles of each process step (i.e. homogenization, centrifugation,washing etc.) for optimal processing. Washing and/or concentrationadjustments using water are typically employed between each processstep/cycle. The pH is adjusted, as needed, for optimal processing. Ingeneral, the following basic processing options may be used to obtain asemi-purified and/or purified inclusion body paste.

The process begins with a fermentation broth comprising a population ofrecombinant microbial host cells comprising insoluble fusion protein inthe form of an inclusion body.

Option 1—Using Initial Cell Separation from Fermentation Broth as aFirst Step.

The fermentation broth is either centrifuged or passed through amembrane filtration process to separate and recover cells containinginclusion bodies of the peptide to be recovered. Water and dissolvedimpurities and salts are removed. The recovered cell mass isre-suspended in water at a concentration of about 10 to about 250 g/Lwet cells. The pH of the mixture is adjusted to a pH of about 9 to about12, more preferentially about 10 to about 11 using a simple strong baselike NaOH. The mixture is then cooled to about 0° to about 10° C. Themixture is passed through a mechanical high pressure homogenizationdevice like a Mouton-Gaulin homogenizer at from about 8,000 psi(approximately 55.2 mPa) to about 25,000 psi (approximately 172 mPa),more preferentially about 10,000 psi (approximately 69.0 mPa) to about15,000 psi (approximately 103 mPa), nominally about 12,000 psi(approximately 82.8 mPa) for several passes. The number of passesthrough the homogenizer may be varied as needed. In one embodiment, thenumber of passes through the homogenizer is about 1 to about 5,preferably 1 to 3, and most preferably about 3. The temperature ofliquid during homogenization is preferably maintained at a temperatureof about 0° C. to about 30° C., preferably about 0° C. to about 10° C.

After the final homogenization pass, the homogenized mixture issubjected to centrifugation and/or filtration. In a preferredembodiment, centrifugation (e.g. stacked disc centrifugation) is used toseparate the insoluble inclusion bodies from the lysate. Theconcentration of lysed cell biomass is optionally adjusted to a lowerconcentration with water prior to centrifugation to 10 to 200 g/L,preferably 50 to 150 g/L, and most preferably about 75 g/L.

Differential settling of the inclusion bodies to a paste occurs and theoverflow of the centrifuge contains the cell debris containing fraction.The recovered inclusion body rich paste is then re-suspended in water.The suspension is well mixed and re-centrifuged or membrane filtered toremove dissolved salts and residual contaminants. If needed, additionalwater washes may be used.

Option 2—Direct Processing of the Fermentation Broth

Direct process of the fermentation broth may also be used. The processis essentially identical to Option 1, except that the fermentation brothis directly processed (no prior centrifugation and/or filtration stepsused to isolate the cells prior to homogenization).

Option 3—The Fermentation Broth is pH Adjusted Before Homogenization

In another embodiment, pH of the fermentation broth may be adjustedprior to homogenization. This option is similar to Option 2, except thatthe pH of the fermentation broth is adjusted to a pH of about 9 to about12, more preferentially about 10 to about 11 prior to homogenization.

High pH Wash Followed by Water Wash

A high pH wash may be used to further purify the inclusion body paste.The concentrated inclusion body paste obtained after centrifugation isadjusted using a 1 M NaHCO₃ pH10 buffer to a final concentration ofabout 50 mM buffer. The suspension is mixed and centrifuged using acentrifuge (e.g. a stacked disk centrifuge) to separate the dissolvedand suspended impurities from the inclusion bodies.

The inclusion body slurry is diluted and washed in water to remove thebuffer. Centrifugation is repeated to isolate the washed inclusion bodypaste.

Cleavage and Oxidative Cross-Linking

In one embodiment, the semi-purified insoluble fusion protein (inclusionbody paste) is re-suspended in water and subjected to a cleavage stepwhereby the fusion protein is cleaved into a mixture of free inclusionbody tag(s), free peptides of interest. The mixture may also includesome partially-cleaved and/or whole fusion proteins. As describedpreviously, the fusion protein comprises one or more cleavable peptidesequences (e.g. cleavable peptide linkers) separating the inclusion bodytags from the peptides of interest. The cleavable peptide linker may becleaved enzymatically and/or chemically (e.g. acid cleavage).

In a preferred embodiment, acid cleavage is used. The inclusion bodyslurry is adjusted to the desired solids concentration (typically about25 g/L on a dry weight basis). The pH of the aqueous solution of fusionpeptides is adjusted so that the acid labile D-P moieties are cleaved. Areducing agent, such as dithiothreitol (DTT, 10 mM) may also be usedduring acid hydrolysis to break disulfide bonds and to promote acidcleavage. Any suitable acid may be used including, but not limited toHCl, formic acid, nitric acid, sulfuric acid, phosphoric acid, citricacid, trifluoroacetic acid, and mixtures thereof. One of skill in theart can adjust the time, temperature, and pH for optimal cleavage.Typically, the acid treatment is conducted at a pH range of about 0.5 toabout 3, more preferably 1.5 to 2.6, most preferably 1.8 to 2.2. Themixture is heated to a temperature of about 40° C. to about 90° C.,preferably 50° C. to about 90° C., more preferably 60° C. to about 80°C., and most preferably about 70° C. The heated acidic mixture is heldfor a period of time from 30 minutes to 48 hours, preferably less than24 hours, even more preferably less than 12 hours, and most preferablyless than 8 hours to achieve effective cleavage.

The cleaved peptide mixture is then cooled to a temperature of about 25°C. and the pH is adjusted to about 5.1 (or the corresponding isoelectricpoint [pI] of the portion containing the plurality of cross-linkablecysteine residues). The pH adjusted solution is further cooled to atemperature of about 0° C. to about 20° C., more preferably about 0° C.to about 10° C., and most preferably about 5° C. and slowly agitatedwith a slow bubbling of filtered air to create an oxidizing environment.The mixture is allowed to cross-link and precipitate for a period oftime sufficient to achieve effective cross-linking. The optimal timerequired for effective cross-linking step can be easily determined byone of skill in the art. Typically, the cross-linking step typicallyranges in time from 5 minutes to about 48 hours, preferably 30 minutesto 24 hours, more preferably about 1 hour to about 12 hours, and mostpreferably about 2 to about 8 hours. The sediment (i.e. the cross-linkedpeptide aggregate) is separated from the supernatant by centrifugationand/or filtration (including microfiltration). The next processing stepis dependent upon which element (i.e. inclusion body tag or peptide ofinterest) was cross-linked:

1. A Cross-Linked Fusion Tag

The isolated supernatant containing the dissolved peptide of interest ispH adjusted as required to precipitate the peptide of interest. Anorganic solvent like acetone, ethanol or methanol may be used to induceprecipitation of the target peptide or impurities. The mixture may becooled to further increase precipitation. The product precipitate isthen recovered by centrifugation or filtration. The precipitate may thenbe washed by chilled solvents or aqueous solvent mixtures. The productmay be dried, re-suspended or dissolved as required for final use.

2. A Cross-Linked Peptide of Interest

The isolated insoluble precipitate (cross-linked peptide of interest)may be further processed into an appropriate product form. In oneembodiment, the isolated precipitate is subjected to reducing conditionsfor a period of time whereby the intermolecular disulfide bonds arebroken. A nitrogen purge and/or a reducing agent such as Na₂SO₃ may beused. Other chemical reducing agents selected from the group consistingof DTT (dithiothreitol), TCEP (Tris(2-carboxyethyl)phosphine),2-mercaptoethanol and 2-mercaptoethylamine. Generally reducing agentsinclude those that contain thiol groups, those that are phosphines andtheir derivatives as well as sulfites and thiosulfites may also be used.In a preferred embodiment, a nitrogen purge is used. The free peptide ofinterest may be subject to additional washing and/or precipitation stepsin order to further purify the material prior to packaging and/or finaluse.

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given either as a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations used is as follows: “min” means minute(s),“h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s),“L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s),“cm” means centimeter(s), “μm” means micrometer(s), “mM” meansmillimolar, “M” means molar, “mmol” means millimole(s), “μmole” meansmicromole(s), “g” means gram(s), “μg” means microgram(s), “mg” meansmilligram(s), “g” means the gravitation constant, “rpm” meansrevolutions per minute, “psi” means pounds per square inch, and “mPa”means megapascal(s).

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or by Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass., 1989. All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), GibcoBRL Life Technologies (Rockville, Md.), Invitrogen (Carlsbad, Calif.) orSigma Aldrich Chemical Company (St. Louis, Mo.), DIFCO Labs (Detroit,Mich.), Promega (Madison, Wis.), QIAgen (Valencia, Calif.), or DNA 2.0Inc. (Menlo Park, Calif.) unless otherwise specified.

Growth Conditions:

E. coli cells were fermented in a 10-L vessel unless otherwise noted.The fermentation proceeded in three stages:

-   -   1. Preparation of 125-mL of seed inoculum. Cells containing the        construct of interest were inoculated in 125-mL of 2YT seed        medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl and        appropriate antibiotic) and grown for several hours at 37° C.    -   2. Growth in batch phase. The 125-mL of inoculum was added to 6        L of batch medium (9 g/L KH₂PO₄, 4 g/L (NH₄)₂HPO₄ 1.2 g/L        MgSO₄.7H₂O, 1.7 g/L citric acid, 5 g/L yeast extract, 0.1 mL/L        Biospumex 153K antifoam, 4.5 mg/L Thiamine.HCl, 23 g/L glucose,        10 mL/L trace elements, 50 mg/L uracil, appropriate antibiotic,        pH 6.7) at 37° C.    -   3. Growth in fed batch phase. After about 12 hours of growth in        the batch phase, the fed-batch phase was initiated. Fed-batch        medium (2 g/L MgSO₄.7H₂O, 4 g/L (NH₄)₂HPO₄ 9 g/L KH₂PO₄, 1-2        g/min Glucose) was added at a constant rate to the reactor for        about 15 hours at 37° C. 4 hours before the end of the fed-batch        phase the cells were induced to express the POI by adding 2 g/L        L-arabinose.

Method to Determine Inclusion Body Formation

To test for the presence of inclusion bodies in the cells, the cellswere lysed with 50 mg of CELLYTIC™ Express (a mixture of non-denaturingdetergents and enzymes available from Sigma, St. Louis, USA) per mL ofgrowth. The inclusion bodies remain insoluble and are spun out with amicro-centrifuge. For large scale isolation after homogenization, astacked-disk centrifugation process was used to isolate the insolubleinclusion bodies.

Example 1 Construction of Expression Plasmids

Several expression systems were used to produce the fusion proteins inan E. coli host cell. One expression system was based on E. coli strainBL21-AI (Invitrogen) in combination with a T7-based expression vector(pLX121; SEQ ID NO: 1; FIG. 1, and pKSIC4-HC7723; FIG. 2; SEQ ID NO: 2)wherein expression of the T7 RNA polymerase is controlled by the araBADpromoter. The other expression system was based on E. coli MG1655 (ATCC46076™) derived strain in combination with a pBAD-based expressionvector (pLR042; FIG. 3; SEQ ID NO: 3, and pLR186; FIG. 4; SEQ ID NO: 4)wherein the endogenous chromosomal copy of the araBAD operon was deleted(the modified E. coli MG1655 strain comprising a disruption in theendogenous araBAD operon is referred to herein as E. coli strainKK2000). The 3′ region downstream and operably linked to the respectivepromoter in each of the vectors was designed to facilitate simpleswapping of the DNA encoding the respective inclusion body tag and/orthe peptide of interest. NdeI and BamHI restriction sites flanked theregion encoding the inclusion body tag (IBT). BamHI and AscI restrictionsites flanked the region encoding the peptide of interest (POI).

The nucleic acid molecules encoding the various fusion peptides weredesigned to include at least one region encoding an inclusion body tag(IBT) linked to a peptide of interest (POI). As described above, thenucleic acid molecules encoding the components of the fusion peptidewere designed to include the appropriate NdeI/BamHI (region encoding theinclusion body tag) and BamHI/AscI restriction sites (region encodingthe peptide of interest) to facilitate insertion in the expressionvector. Insertion of the nucleic acid molecules created a chimeric geneencoding a fusion peptide operably linked to the respective promoter.The fusion peptide was designed to have an inclusion body tag (IBT)linked to a peptide of interest (POI) where the two components wereseparated by a cleavable peptide linker (CS; for example, an acidcleavable DP moiety):

Construction of pLX121 Expression Plasmid (T7-Based Expression):

A genetic construct was prepared for evaluating the performance of thecross-linkable inclusion body tags when fused to a soluble peptide ofinterest. A plasmid (pLX121; FIG. 1; SEQ ID NO: 1) containing a pBR322origin of replication and the bla gene to confer ampicillin resistancewas used. Expression of the chimeric gene was driven by a T7 promoter.Construction of this plasmid is previously described in co-pending U.S.patent application Ser. No. 11/516,362, herein incorporated byreference.

Briefly, the pLX121 expression vector was designed from the destinationplasmid pDEST17 (Invitrogen. Carlsbad, Calif.). The expression vectorwas modified so that the chimeric gene encoding the fusion protein wasexpressed under the control of the T7 promoter. NdeI and BamHIrestriction sites were used for easy swapping of the various inclusionbody tags. BamHI and AscI restriction sites were used to facilitateswapping of various peptides of interest. The sequence encoding thejunction between the inclusion body tag and the peptide of interest wasdesigned to encode an acid cleavable D-P moiety.

Construction of Expression Vector pKSIC4-HC77623

The vector pKSIC4-HC77623 (SEQ ID NO: 2; FIG. 2) was also derived fromthe commercially available vector pDEST17 (Invitrogen). Construction ofthis vector has been previously described in co-pending U.S. patentapplication Ser. No. 11/389,948, herein incorporated by reference. Itincludes sequences derived from the commercially available vector pET31b(Novagen, Madison, Wis.) that encode a fragment of the enzymeketosteroid isomerase (KSI; Kuliopulos, A. and Walsh, C. T., J. Am.Chem. Soc. 116:4599-4607 (1994)). The KSI fragment used as an inclusionbody tag to promote partition of the peptides into insoluble inclusionbodies in E. coli. The nucleic acid molecule encoding the KSI sequencefrom pET31 b was modified using standard mutagenesis procedures(QuickChange II, Stratagene, La Jolla, Calif.) to include threeadditional cysteine codons, in addition to the one cysteine codon foundin the wild type KSI sequence, resulting in the inclusion body tag KSI4C(SEQ ID NOs: 5 and 6). The plasmid pKSIC4-HC77623 was constructed usingstandard recombinant DNA methods well known to those skilled in the art.The BamHI and AscI restriction sites facilitated swapping of nucleicacid molecules encoding the various peptides of interest. The insertswere designed to encode an acid cleavable DP moiety useful in separatingthe inclusion body tag from the peptide of interest.

Construction of pLR042 Expression Plasmid (pBAD Based Expression)

Plasmid pLR042 (SEQ ID NO: 3; FIG. 3) contains a ColE1 type origin ofreplication, the bla gene to confer ampicillin resistance and the aadA-1gene to confer spectinomycin (Spec) resistance. The tag/peptide fusionconstruct is driven by the pBAD promoter. The plasmid also encodes thegene for the araC regulator.

Plasmid pLR042 was derived from the commercially available plasmidpBAD-HisA (Invitrogen). Briefly, a modified multiple cloning site (MCS)was cloned in pBAD-HisA and the NdeI restriction site at position 2844was removed to create a single NdeI site downstream of the pBADpromoter. The resulting plasmid was named pBAD-HisA_MCSmod. TheNdeI/EcoRI fragment of plasmid pKSIC4-HC77623 was inserted into theNdeI/EcoRI site of pBAD-HisA_MCSmod, creating plasmidpSF004_pBAD-KSIC4-HC77623. The HindIII fragment of plasmid pCL1920(Lerner and Inouye, Nucleic Acids Research, 18:4631 (1990); GENBANK®Accession No. AB236930) comprising the spectinomycin resistance gene(aadA-1) was inserted into pSF004_pBAD-KSI4-HC77623, creating plasmidpLR042 (FIG. 4; SEQ ID NO: 3).

Construction of pLR186 Expression Plasmid:

Plasmid pLR186 (FIG. 4; SEQ ID NO: 4) was created from plasmid pLR042(SEQ ID NO: 3; FIG. 3) by removing the coding region for theKSIC4-HC77623 fusion peptide and inserting the coding region for fusionpeptide IBT139-HC776124 (i.e. a fusion peptide comprising inclusion bodytag IBT-139 linked to the HC776124 peptide of interest; see Example 5).

Example 2 KSI Inclusion Body Tag without an Effective Number ofCross-Linkable Cysteines Cannot be Easily Separated from the CleavedPeptide by Simple Physical Methods

The purpose of this example is to show that separation of the inclusionbody tag and peptide is more difficult if the tag is not selectivelycross-linked via cysteines and subsequently precipitated. In thisexample the peptide and IB-tag were separated using preparative HPLC.

Construct: KSI.HC77607 (SEQ ID NOs: 7 and 8; Table 2). Peptide HC77607does have cysteine residues, however, in this example it was not used asa separation tool (Table 2). Peptide HC77607 (i.e. the peptide ofinterest) is comprised of several hair binding domains (bold) includingKF11 (SEQ ID NO: 9) and D21′ (RTNAADHP; SEQ ID NO: 10). The acidcleavable DP moiety is italicized.

TABLE 2 Components of hair binding peptide HC77607 Nucleic Amino acidAcid Peptide Amino acid SEQ ID SEQ ID Name Formula Sequence NO: NO:HC77607 GSDP-KF11-GGG- GSDPNTSQLSTGGG 11 12 D21′-KCGGG-KF11-RTNAADHPKCGGGN GGG-D21′-KCGGG- TSQLSTGGGRTNAA KF11-GGG-D21′-KCDHPKCGGGNTSQLS TGGGRTNAADHPKCCloning of KSI-HC77607: The genes for KSI and HC77607 were synthesizedby DNA2.0 (Menlo Park, Calif.) with appropriate restriction sites andcloned into pLX121 as described above.Growth Conditions: Growth and expression of the chimeric gene encodingthe fusion peptide was conducted as described above.

Isolation of Fusion Protein and HPLC Analysis:

The whole fermentation broth was passed through an APV model 2000 Gaulintype homogenizer at 12,000 psi (82,700 kPa) for three passes. The brothwas cooled to below 5° C. prior to each homogenization. The homogenizedbroth was immediately processed through a Westfalia WHISPERFUGE™(Westfalia Separator Inc., Northvale, N.J.) stacked disc centrifuge at600 mL/min and 12,000 relative centrifugal force (RCF) to separateinclusion bodies from suspended cell debris and dissolved impurities.The recovered paste was re-suspended at 15 g/L (dry basis) in water andthe pH adjusted to about 10.0 using NaOH. The suspension was passedthrough the APV 2000 Gaulin type homogenizer at 12,000 psi (82,700 kPa)for a single pass to provide rigorous mixing. The homogenized pH 10suspension was immediately processed in a Westfalia WHISPERFUGE™ stackeddisc centrifuge at 600 mL/min and 12,000 RCF to separate the washedInclusion bodies from suspended cell debris and dissolved impurities.The recovered paste was resuspended at 15 gm/L (dry basis) in purewater. The suspension was passed through the APV 2000 Gaulin typehomogenizer at 12,000 psi (82,700 kPa) for a single pass to providerigorous washing. The homogenized suspension was immediately processedin a Westfalia WHISPERFUGE™ stacked disc centrifuge at 600 mL/min and12,000 RCF to separate the washed Inclusion bodies from residualsuspended cell debris and NaOH. The recovered paste was resuspended inpure water at 25 gm/L (dry basis) and the pH or the mixture adjusted to2.2 using HCl. Dithiothreitol (DTT) was added to 10 mM (when processingthe HC77607 peptide). The acidified suspension was heated to 70° C. for14 hours to complete cleavage of the DP site separating the fusionpeptide from the product peptide. The product was pH neutralized (note:the pH used may vary depending upon the solubility of the peptide beingrecovered) and cooled to ˜5° C. and held for 12 hours. During this stepthe suspension was held in a 500-mL or 1-L bottle no more than ¾ full toensure adequate presence of oxygen to ensure cysteine cross linkingthrough disulfide formation. The mixture was then centrifuged at 9000RCF for 30 minutes and the supernatant decanted for HPLC analysis.

HPLC Method

The supernatant was filtered with a 0.2 micron membrane. The filteredproduct was loaded in a 22×250 mm reverse phase chromatography columnGRACEVYDAC® (218TP1022) containing 10 micron C18 media which waspreconditioned with 10% acetonitrile (ACN), 90% water with 0.1% v/vtrifluoroacetic acid (TFA). The product was recovered in a purifiedstate by eluting the column with a gradient of water and acetonitrile(ACN) ramping from 10% to 25% acetonitrile (ACN) in water with TFA at0.1% v/v at room temperature and approximately 10 mL/min.Spectrophotometric detection at 220 nm was used to monitor and trackelution of the product peptide.

Result:

The solubility tag and peptide were separated using the preparative HPLCmethod described in above. The IBTs and POIs were both found in thesupernatant.

Example 3 An Inclusion Body Tag KSI(C4) with an Effective Number ofCross-Linkable Cysteines is Easily Separated from a Cleaved PeptideMixture by Precipitation

The purpose of this example is to show that separation of the IBT andpeptide of interest can by achieved by oxidatively cross-linking thecysteine residues within the IBT and subsequent precipitation of thetag. The peptide of interest was HC77643 (contains no cysteineresidues). The remaining soluble peptide was shown to be free of theKSI(C4) tag by using HPLC.

Construct: KSI(C4).HC77643 (SEQ ID NOs: 13 and 14)

The design of peptide HC77643 is provided in Table 3 Peptide HC77643 iscomprised of several hair binding domains including A09 (SEQ ID NO: 15)and KF11 (SEQ ID NO: 9) (bold). The acid cleavable DP moiety isitalicized.

TABLE 3 Components of Multi-block Hair-binding Peptide HC77643 NucleicAmino Peptide Amino acid acid Acid Name Formula Sequence SEQ ID NO:SEQ ID NO: HC77643 DPG-A09-GAG- DPGIPWWNIRAPLNA 16 17 A09-GGSGPGSGG-GAGIPWWNIRAPLNA KF11-GGG-KF11- GGSGPGSGGNTSQL GGPKK STGGGNTSQLSTGG PKKCloning of KSI(C4).HC77643: The genes for KSI(C4) (SEQ ID NO: 5) andHC77643 (SEQ ID NO:16) were synthesized by DNA2.0 (Menlo Park, Calif.)with appropriate restriction sites and cloned into pLX121 as mentionedabove.

Production of Product Protein:

Growth and expression of the chimeric gene were conducted as describedabove. The protein was purified as described in above. After the acidcleavage and pH neutralization, the mixture was stored at approximately5° C. for about 6 hours to allow the cysteines to form cross-linkedbonds. Oxygen to drive the cysteine cross-linking was provided by a 30%bottle air volume. The mixture was centrifuged at 9000 RCF for 30minutes and the precipitated tag was separated from the soluble peptide.

Results:

SDS-PAGE gel analysis of both the precipitated paste (comprised ofcross-linked IBTs) and the remaining soluble fraction showed thepresence of KSI(C4) in the insoluble paste, and HC77643 remaining in thesoluble fraction. This was further confirmed by HPLC (using the HPLCmethod described in Example 2), which showed only the presence ofHC77643 in the soluble fraction. The results of the cross-linkingexperiments are summarized in Table 5.

Example 4 Small Inclusion Body Tag (IBT139) without Cysteines

The large KSI tag used in the previous examples is effective in inducinginclusion body formation. However, the use of a smaller IBT increasesthe relative yield of the peptide of interest when prepared as a fusionpeptide. The purpose of this example is to show that a small inclusionbody tag (for example, a small inclusion body tag herein referred to asIBT139; SEQ ID NO: 18) can drive the fusion peptides into inclusionbodies.

Construct: IBT139.HC776124 (pLR186) (SEQ ID NOs: 19 and 20). The designof peptide HC776124 is provided in Table 4. Peptide HC776124 (a dimer ofHC77643) is comprised of several hair binding domains including A09 (SEQID NO: 15) and KF11 (SEQ ID NO: 9) (bold). The acid cleavable DPmoieties are italicized (Table 4).

TABLE 4 Nucleic Amino Acid Acid Peptide Amino acid SEQ ID SEQ ID NameFormula Sequence NO: NO: HC776124 D(PG-A09-GAG- DPGIPWWNIRAPLNAGAGIP 2122 A09- WWNIRAPLNAGGSGPGSGG GGSGPGSGG- NTSQLSTGGGNTSQLSTGGPKF11-GGG-KF11- KKPGDPGIPWWNIRAPLNAG GGPKKPGD)2 AGIPWWNIRAPLNAGGSGPGSGGNTSQLSTGGGNTSQLST GGPKKPGD

Cloning and Initial Analysis of IBT139.HC776124:

A 56 amino acid tag IBT139 (SEQ ID NO: 18), was identified as beingeffective in driving the fusion peptides into inclusion bodies. HC776124(i.e. the POI) was synthesized by DNA2.0 (Menlo Park, Calif.) and clonedinto restriction sites BamHI (5′) and AscI (3′) of plasmid pLR042 (seeExample 1). The resulting plasmid was designated as pLR186 (FIG. 2; SEQID NO: 4).

The pLR186 construct was transformed into E. coli MG1655 (ATCC 46076™)with the endogenous chromosomal araBAD operon deleted. A 3-mL growth inLB (plus 100 μg/mL of ampicillin) was inoculated with 30 μL of anovernight culture. The culture was grown to OD₆₀₀ of about 0.4 andinduced with 0.2% arabinose and grown for 3 hours. To determine solubleversus insoluble cell content, the cells were lysed and soluble andinsoluble fractions were run on an SDS-PAGE gel. The fusion proteinproduced was made in the form of insoluble inclusion bodies.

Production of Product Protein:

The fusion protein was produced and processed as described above.

Results:

IBT139 was effective in promoting inclusion body formation.

Example 5 Small Inclusion Body Tag (IBT186) Comprising an EffectiveAmount of Cross-Linkable Cysteines can be Separated from the CleavedPeptide Mixture by Oxidative Cross-Linking and Precipitation

The purpose of this example is to show that a small tag inclusion bodytag (e.g. IBT186; SEQ ID NOs: 23 and 24) containing an effective numberof cross-linkable cysteine residues (IBT186 contains 4 cysteineresidues) can drive both inclusion body formation while being easy toseparate using oxidative cross-linking. The example also shows that asmall inclusion body tag previous shown to be effective in inducinginclusion body formation can be modified to contain an effective amountof cross-linkable cysteine residues (IBT186 is derived from small tagIBT139 (Example 4) with four cysteines distributed within its sequence)while maintaining its ability to effectively drive inclusion bodyformation. The presence of four cysteines allows simple precipitation ofthe tag after cleavage of tag and peptide.

Construct: IBT186-HC776124 (pLR238) (SEQ ID NOs: 25 and 26)

Cloning and Initial Analysis of IBT186.HC776124:

The coding sequence (SEQ ID NO: 23) encoding IBT186 was synthesized byDNA2.0 (Menlo Park, Calif.) and cloned into restriction sites NdeI (5′)and BamHI (3′) of plasmid pLR186 (expression driven off pBAD promoter)to make a fusion with the HC776124 construct, creating plasmid pLR238.The plasmid was transformed into E. coli MG1655 (ATCC 46076™) with thearaBAD operon deleted.

A 3-mL growth in LB (plus 100 μg/mL of ampicillin) was inoculated with30 μL of an overnight culture. The culture was grown to OD₆₀₀ of about0.4 and induced with 0.2% arabinose and grown for 3 hours. To determinesoluble versus insoluble cell content, the cells were lysed and solubleand insoluble fractions were run on an SDS-PAGE gel. The fusion proteinproduced was again made as insoluble inclusion bodies.

Production of Product Protein:

The protein was produced and processed as described above. After theacid cleavage and pH neutralization, the mixture was stored at ˜5° C.for about 6 hours to allow the cysteines to form cross-linked bonds.Ambient air exposure provided oxygen to cause cysteine cross-linking.The mixture was centrifuged at 9000 RCF for 30 minutes and theprecipitated inclusion body tag was separated from the soluble peptideof interest.

Results:

SDS-PAGE gel analysis of both the precipitate paste and the remainingsoluble fraction showed the presence of IBT186 in the insoluble pasteand HC776124 remaining in the soluble fraction. This was furtherconfirmed by HPLC (see method described in Example 2), which showed onlythe presence of HC776124 in the soluble fraction. The results of thecross-linking experiments are summarized in Table 5.

Example 6 Small Inclusion Body Tag IBT139(5C) Comprising an EffectiveAmount of Cross-Linkable Cysteines can be Separated from the CleavedPeptide Mixture by Oxidative Cross-Linking and Precipitation

The purpose of this example is to show that another small tag inclusionbody tag (e.g. IBT139(5C); SEQ ID NOs: 181-192) containing an effectivenumber of cross-linkable cysteine residues (IBT139(5C) contains 5cysteine residues) can drive both inclusion body formation while beingeasy to separate using oxidative cross-linking. The example also showsthat a small inclusion body tag previous shown to be effective ininducing inclusion body formation can be modified to contain aneffective amount of cross-linkable cysteine residues (IBT139(5C) isderived from small tag IBT139 (Example 4) with five cysteinesdistributed within its sequence) while maintaining its ability toeffectively drive inclusion body formation. The presence of fivecysteines allows simple precipitation of the tag after cleavage of tagand peptide of interest.

Construct: IBT139(5C)-HC776124 (pLR435) (SEQ ID NOs: 183-184)

Cloning and Initial Analysis of IBT139(5C).HC776124:

The coding sequence (SEQ ID NO: 181) encoding IBT139(5C) (SEQ ID NO:182) was synthesized by DNA2.0 (Menlo Park, Calif.) and cloned intorestriction sites NdeI (5′) and BamHI (3′) of plasmid pLR186 (expressiondriven off pBAD promoter) to make a fusion with the HC776124 (SEQ ID NO:22) construct, creating plasmid pLR435 (SEQ ID NO: 180). The plasmid wastransformed into E. coli MG1655 (ATCC 46076™) with the native araBADoperon deleted. The sequence of IBT139(5C) comprising the 5 cysteineresidues (bold) is provided below.

IBT139(5C):

(SEQ ID NO: 182) MASCGQQRFQWQFEQQPRCGQQRFQWQFEQQPRCGQQRFQWQFEQQPECGQQRFQWQFEQQPC.

A 3-mL growth in LB (plus 100 μg/mL of ampicillin) was inoculated with30 μL of an overnight culture. The culture was grown to OD₆₀₀ of about0.4 and induced with 0.2% arabinose and grown for 3 hours. To determinesoluble versus insoluble cell content, the cells were lysed and solubleand insoluble fractions were run on an SDS-PAGE gel. The fusion proteinproduced was again made as insoluble inclusion bodies.

Production of Product Protein:

The protein was produced and processed as described above. After theacid cleavage and pH neutralization, the mixture was stored at ˜5° C.for about 6 hours to allow the cysteine residues to oxidize and formcross-linked bonds. Ambient air exposure provided sufficient oxygen tocause cysteine cross-linking. The mixture was subsequently centrifugedat 9000 RCF for 30 minutes and the precipitated inclusion body tag wasseparated from the soluble peptide of interest.

Results:

SDS-PAGE gel analysis of both the precipitate paste and the remainingsoluble fraction showed the presence of IBT139(5C) in the insolublepaste and HC776124 remaining in the soluble fraction. This was furtherconfirmed by HPLC (see method described in Example 2), which showed onlythe presence of HC776124 in the soluble fraction. The results of thecross-linking experiments are summarized in Table 5.

Example 7 Introduction of Multiple Cysteines to the Terminus of anInclusion Body Tag Promotes Oxidative Cross-Linking while Retaining theAbility to Effectively Drive Fusion Peptides into Inclusion Bodies

The purpose of this example is to show that the addition of across-linkable cysteine motif comprising effective number of cysteineresidues to the terminus of an inclusion body tag creates across-linkable IBT, even when the cysteines are spaced closely together.A cross-linkable cysteine motif was added to an inclusion body tagnormally devoid of cross-linkable cysteine residues (i.e. IBT139; SEQ IDNO: 18), creating cysteine modified tag “IBT139.CCPGCC” (SEQ ID NO: 27).The addition of the motif did not alter the IBT's ability to driveinclusion body formation while the modification facilitated simpleseparation of the tag using oxidative cross-linking. The results of thecross-linking experiments are summarized in Table 5.

Construct: IBT139.CCPGCC. HC776124 (SEQ ID NOs: 28 and 29). Cloning andInitial Analysis:

To facilitate crosslinking, the tetracysteine tag CCPGCC (SEQ ID NO: 31)was introduced at the end of the inclusion body promoting sequenceIBT139 (SEQ ID NO: 18) which does not naturally contain cysteineresidues. The CCPGCC tetracysteine tag is the LUMIO™ biarsenical dyebinding motif. The LUMIO™ Green detection kit was obtained fromInvitrogen (Invitrogen, Carlsbad, Calif.)The oligonucleotides encoding the tetracysteine tag CCPGCC (SEQ IDNO:30) were synthesized by Sigma Genosys. The top strand oligo5′-GATCTTGCTGTCCGGGCTGTTGCG-3′ (SEQ ID NO: 32) and the bottom strandoligo 5′-GATCCGCAACAGCCCGGACAGCAA-3′ (SEQ ID NO: 33) were annealed witha BglII overhang at the 5′ end and a BamHI overhang at the 3′ end. Theannealed double stranded fragment was cloned into the BamHI site of apeptide expression plasmid pLR186, creating plasmid pLR199. PlasmidpLR199 contained the peptide of interest HC776124 fused to the inclusionbody promoting sequence IBT139 expressed by the P_(BAD) promoter. Theresulting clone contained the tetracysteine tag CCPGCC (SEQ ID NO: 31)inserted after the inclusion body promoting sequence and before the acidcleavage site. It was shown that the introduction of the tetracysteinemoiety did not affect expression or localization of the peptides byrunning an equivalent number of cells on a protein gel and seeing samelevels of expression. The overexpressed protein was shown to be in theform of inclusion bodies by treating the cells with CELLYTIC™ Expressand verifying that they were in the insoluble fraction. The inclusionbody tag promoting sequence IBT139 with addition of the cross-linkableCCPGCC tag did not alter the inclusiobn body tag's ability to forminclusion bodies (Table 5).

Production of Product Protein:

The protein was produced and processed as described above. After theacid cleavage and pH neutralization, the mixture was stored at ˜5° C.for at least 6 hours to allow the cysteines to form cross-linked bonds.Ambient air exposure provided oxygen to cause cysteine cross-linking.The mixture was centrifuged at 9000 RCF for 30 minutes and theprecipitated tag was separated from the soluble peptide.

Results:

SDS-PAGE gel analysis of both the precipitated paste and the remainingsoluble fraction showed the presence of the inclusion body tag(IBT139.CCPGCC) in the insoluble paste and the peptide of interest(HC776124) remaining in the soluble fraction. This was further confirmedby HPLC analysis (see Example 2), which showed only the presence ofHC776124 in the soluble fraction. The results of the cross-linkingexperiments are summarized in Table 5.

TABLE 5 Summary of Cross-Linking Results Number of IBT CysteinesSeparation Induces IB in the via Oxidative Formation inclusionCross-linking and Construct Evaluated in Cell body tag CentrifugationKSI.HC77607 Yes None No KSI(C4).HC77643 Yes 4 Yes IBT139.HC776124 YesNone No IBT186.HC776124 Yes 4 Yes IBT139.CCPGCC.HC776124 Yes 4 YesIBT139(5C).HC776124 Yes 5 Yes

1-2. (canceled)
 3. A process to obtain a peptide of interest from a fusion peptide comprising: a) providing a population of fusion peptides comprising the general structure: IBT-CS-POI or POI-CS-IBT wherein; i) IBT is an inclusion body tag that does not include a cysteine residue; ii) CS is a cleavage site; and iii) POI is a peptide of interest comprising an effective number of cysteine residues; b) cleaving the population of fusion peptides at said cleavage site whereby the inclusion body tag is no longer linked to the peptide of interest and whereby a mixture of peptide molecules is produced comprising a plurality of inclusion body tags and a plurality of peptides of interest; c) subjecting the mixture of peptide molecules of step (b) to oxidizing conditions whereby the peptides of interest are cross-linked; and d) recovering the peptide of interest.
 4. A process according to claim 3 wherein the population of fusion peptides is produced in a recombinant host cell comprising a nucleic acid molecule encoding said fusion peptides.
 5. The process of claim 3 wherein the effective number of cysteine residues in the peptide of interest is at least
 3. 6. (canceled)
 7. The process of claim 3 wherein the population of fusion peptides is cleaved by a cleavage reagent selected from the group consisting of chemical cleavage reagents and enzymatic cleavage reagents.
 8. The process of claim 7 wherein the chemical cleavage reagent is an effective amount of an acid cleavage reagent.
 9. The process of claim 3 wherein inclusion body tag is less than 125 amino acids in length.
 10. The process of claim 3 wherein the peptide of interest is less than 300 amino acids in length.
 11. The process of claim 10 wherein the peptide of interest is selected from the group consisting of body surface-binding peptides, hair-binding peptides, skin-binding peptides, nail-binding peptides, teeth-binding peptides, cellulose-binding peptides, polymer-binding peptides, clay-binding peptides, pigment-binding peptides, and antimicrobial peptides.
 12. The process of claim 4 wherein the recombinant host cell is a microbial host cell.
 13. The process of claim 12 wherein the microbial host cell is selected from the group consisting of bacteria, fungi, and yeast.
 14. The process of claim 13 wherein the microbial host cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus.
 15. The process of claim 3 wherein the oxidizing conditions comprises the presence of a gas comprising an effective amount of diatomic or triatomic oxygen. 